@article {5388, title = {Occupational cosmic radiation exposure in Portuguese airline pilots: study of a possible correlation with oxidative biological markers.}, journal = {Radiation and environmental biophysics}, volume = {52}, number = {2}, year = {2013}, note = {n/a}, month = {Jun}, pages = {211-220}, abstract = {

Several studies have sought to understand the health effects of occupational exposure to cosmic radiation. However, only few biologic markers or associations with disease outcomes have so far been identified. In the present study, 22 long- and 26 medium-haul male Portuguese airline pilots and 36 factory workers who did not fly regularly were investigated. The two groups were comparable in age and diet, were non-smokers, never treated with ionizing radiation and other factors. Cosmic radiation exposure in pilots was quantified based on direct monitoring of 51 flights within Europe, and from Europe to North and South America, and to Africa. Indirect dose estimates in pilots were performed based on the SIEVERT (Syst{\`e}me informatis{\'e} d\'{\'e}valuation par vol de l\'exposition au rayonnement cosmique dans les transports a{\'e}riens) software for 6,039 medium- and 1,366 long-haul flights. Medium-haul pilots had a higher cosmic radiation dose rate than long-haul pilots, that is, 3.3~{\textpm}~0.2 μSv/h and 2.7~{\textpm}~0.3 μSv/h, respectively. Biological tests for oxidative stress on blood and urine, as appropriate, at two time periods separated by 1~year, included measurements of antioxidant capacity, total protein, ferritin, hemoglobin, creatinine and 8-hydroxy-2-deoxyguanosine (8OHdG). Principal components analysis was used to discriminate between the exposed and unexposed groups based on all the biological tests. According to this analysis, creatinine and 8OHdG levels were different for the pilots and the unexposed group, but no distinctions could be made among the medium- and the long-haul pilots. While hemoglobin levels seem to be comparable between the studied groups, they were directly correlated with ferritin values, which were lower for the airline pilots.

}, url = {http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed\&id=23412012\&retmode=ref\&cmd=prlinks}, author = {Silva, Rodrigo and Folgosa, Filipe and Soares, Paulo and Pereira, Alice S. and Garcia, Raquel and Gestal-Otero, Juan Jesus and Tavares, Pedro and Gomes da Silva, Marco D R} } @article {5333, title = {Bacterioferritin from Desulfovibrio vulgaris Hildenborough is a functional DPS-like enzyme}, journal = {FEBS JOURNAL}, volume = {{279}}, number = {{1, SI}}, year = {2012}, note = {

22nd IUBMB Congress/37th FEBS Congress, Seville, SPAIN, SEP 04-09, 2012

}, month = {Sep}, pages = {{465}}, abstract = {n/a}, author = {Folgosa, F. and Tim{\'o}teo, C G and Guilherme, M. and Penas, D. and Tavares, P and Pereira, A. S.} } @article {5318, title = {Synthesis of catecholamine conjugates with nitrogen-centered bionucleophiles}, journal = {BIOORGANIC CHEMISTRY}, volume = {{44}}, year = {2012}, note = {n/a}, month = {Oct}, pages = {{19-24}}, publisher = {ACADEMIC PRESS INC ELSEVIER SCIENCE}, address = {525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USA}, abstract = {

The enzymatic (tyrosinase) and chemical (NaIO4, Ag2O or Fremys\'s salt) oxidation of biologically relevant catecholamines, such as dopamine (DA), N-acetyldopamine (NADA) and the Ecstasy metabolites (alpha-MeDA and N-Me-alpha-MeDA) generates the corresponding o-quinone which can be trapped with nitrogen bionucleophiles such as N-acetyl-histidine and imidazole in a regioselective reaction that takes place predominantly at the 6-position of the catecholamine. (C) 2012 Elsevier Inc. All rights reserved.

}, author = {Siopa, Filipa and Pereira, Alice S. and Ferreira, Luisa M. and Matilde Marques, M. and Branco, Paula S.} } @article {5332, title = {Desulfovibrio vulgaris bacterioferritin uses H2O2 as a co-substrate for iron oxidation and reveals DPS-like DNA protection and binding activities}, journal = {BIOCHEMICAL JOURNAL}, volume = {{446}}, number = {{1}}, year = {2012}, note = {n/a}, pages = {{125-133}}, abstract = {

A gene encoding Bfr (bacterioferritin) was identified and isolated from the genome of Desulfovibrio vulgaris cells, and overexpressed in Escherichia coli. In vitro, H2O2 oxidizes Fe2+ ions at much higher reaction rates than O-2. The H2O2 oxidation of two Fe2+ ions was proven by Mossbauer spectroscopy of rapid freeze-quenched samples. On the basis of the Mossbauer parameters of the intermediate species we propose that D. vulgaris Bfr follows a mineralization mechanism similar to the one reported for vertebrate H-type ferritins subunits, in which a diferrous centre at the ferroxidase site is oxidized to diferric intermediate species, that are subsequently translocated into the inner nanocavity. D. vulgaris recombinant Bfr oxidizes and stores up to 600 iron atoms per protein. This Bfr is able to bind DNA and protect it against hydroxyl radical and DNase deleterious effects. The use of H2O2 as an oxidant, combined with the DNA binding and protection activities, seems to indicate a DPS (DNA-binding protein from starved cells)-like role for D. vulgaris Bfr.

}, author = {Timoteo, Cristina G. and Guilherme, Marcia and Penas, Daniela and Folgosa, Filipe and Tavares, Pedro and Pereira, Alice S.} } @article {5334, title = {Spectroscopic Evidence for and Characterization of a Trinuclear Ferroxidase Center in Bacterial Ferritin from Desulfovibrio vulgaris Hildenborough}, journal = {Journal Of The American Chemical Society}, volume = {{134}}, number = {{26}}, year = {2012}, note = {n/a}, pages = {{10822-10832}}, abstract = {

Ferritins are ubiquitous and can be found in practically all organisms that utilize Fe. They are composed of 24 subunits forming a hollow sphere with an inner cavity of similar to 80 angstrom in diameter. The main function of ferritin is to oxidize the cytotoxic Fe2+ ions and store the oxidized Fe in the inner cavity. It has been established that the initial step of rapid oxidation of Fe2+ (ferroxidation) by H-type ferritins, found in vertebrates, occurs at a diiron binding center, termed the ferroxidase center. In bacterial ferritins, however, X-ray crystallographic evidence and amino acid sequence analysis revealed a trinuclear Fe binding center comprising a binuclear Fe binding center (sites A and B), homologous to the ferroxidase center of H-type ferritin, and an adjacent mononuclear Fe binding site (site C). In an effort to obtain further evidence supporting the presence of a trinuclear Fe binding center in bacterial ferritins and to gain information on the states of the iron bound to the trinuclear center, bacterial ferritin from Desulfovibrio vulgaris (DvFtn) and its E130A variant was loaded with substoichiometric amounts of Fe2+, and the products were characterized by Mossbauer and EPR spectroscopy. Four distinct Fe species were identified: a paramagnetic diferrous species, a diamagnetic diferrous species, a mixed valence Fe2+Fe3+ species, and a mononuclear Fe2+ species. The latter three species were detected in the wild-type DvFtn, while the paramagnetic diferrous species was detected in the E130A variant. These observations can be rationally explained by the presence of a trinuclear Fe binding center, and the four Fe species can be properly assigned to the three Fe binding sites. Further, our spectroscopic data suggest that (1) the fully occupied trinuclear center supports an all ferrous state, (2) sites B and C are bridged by a mu-OH group forming a diiron subcenter within the trinuclear center, and (3) this subcenter can afford both a mixed valence Fe2+Fe3+ state and a diferrous state. Mechanistic insights provided by these new findings are discussed and a minimal mechanistic scheme involving O-O bond cleavage is proposed.

}, author = {Pereira, Alice S. and Timoteo, Cristina G. and Guilherme, Marcia and Folgosa, Filipe and Naik, Sunil G. and Duarte, Americo G. and Huynh, Boi Hanh and Tavares, Pedro} } @article {5387, title = {Comparative electrochemical study of superoxide reductases}, journal = {Eur. Biophys. J.}, volume = {41}, number = {2}, year = {2011}, note = {n/a}, month = {Dec 06}, pages = {209-215}, abstract = {

... CM Cordas (\&) {\'A} P . Raleiras {\'A} F . Auche{\textquoteleft}re {\'A} I. Moura {\'A} JJG Moura ... de Qu{\i}mica, Universidade Nova de Lisboa, 2859-516 Caparica, Portugal e-mail: cristina. cordas @dq.fct ... Present Address: P . Raleiras Department of Photochemistry and Molecular Science, PO Box 523, 75120 ...

}, url = {http://www.springerlink.com/index/10.1007/s00249-011-0777-1}, author = {Cordas, Cristina M. and Raleiras, Patricia and Auch{\`e}re, Fran{\c c}oise and Moura, Isabel and Moura, Jose J. G.} } @article {5336, title = {Electrochemical behaviour of Dps-a mini-ferritin}, journal = {EUROPEAN BIOPHYSICS JOURNAL WITH BIOPHYSICS LETTERS}, volume = {{40}}, number = {{1}}, year = {2011}, note = {

8th EBSA European Biophysics Congress, Budapest, HUNGARY, AUG 23-27, 2011

}, month = {Aug}, pages = {{181}}, abstract = {n/a}, author = {Cordas, Cristina M. and Wilton, Joana and Cardoso, Tania and Folgosa, Filipe and Pereira, Alice S. and Tavares, Pedro} } @article {5337, title = {Low-Spin Heme b(3) in the Catalytic Center of Nitric Oxide Reductase from Pseudomonas nautica}, journal = {Biochemistry}, volume = {{50}}, number = {{20}}, year = {2011}, note = {n/a}, pages = {{4251-4262}}, abstract = {

Respiratory nitric oxide reductase (NOR) was purified from membrane extract of Pseudomonas (Ps.) nautica cells to homogeneity as judged by polyacrylamide gel electrophoresis. The purified protein is a heterodimer with subunits of molecular masses of 54 and 18 kDa. The gene encoding both subunits was cloned and sequenced. The amino acid sequence shows strong homology with enzymes of the cNOR class. Iron/heme determinations show that one heme c is present in the small subunit (NORC) and that approximately two heme b and one non-heme iron are associated with the large subunit (NORB), in agreement with the available data for enzymes of the cNOR class. Mossbauer characterization of the as-purified, ascorbate-reduced, and dithionite-reduced enzyme confirms the presence of three heme groups (the catalytic heme b(3) and the electron transfer heme b and heme c) and one redox-active non-heme Fe (Fe-B). Consistent with results obtained for other cNORs, heme c and heme b in Ps. nautica cNOR were found to be low-spin while FeB was found to be high-spin. Unexpectedly, as opposed to the presumed high-spin state for heme b(3), the Mossbauer data demonstrate unambiguously that heme b(3) is, in fact, low-spin in both ferric and ferrous states, suggesting that heme b(3) is six-coordinated regardless of its oxidation state. EPR spectroscopic measurements of the as-purified enzyme show resonances at the g similar to 6 and g similar to 2-3 regions very similar to those reported previously for other cNORs. The signals at g = 3.60, 2.99, 2.26, and 1.43 are attributed to the two charge-transfer low-spin ferric heme c and heme b. Previously, resonances at the g similar to 6 region were assigned to a small quantity of uncoupled high-spin Fe-III heme b(3). This assignment is now questionable because heme b(3) is low-spin. On the basis of our spectroscopic data, we argue that the g = 6.34 signal is likely arising from a spin spin coupled binuclear center comprising the low-spin Fe-III heme b(3) and the high-spin Fe-B(III). Activity assays performed under various reducing conditions indicate that heme b(3) has to be reduced for the enzyme to be active. But, from an energetic point of view, the formation of a ferrous heme-NO as an initial reaction intermediate for NO reduction is disfavored because heme [FeNO](7) is a stable product. We suspect that the presence of a sixth ligand in the Fe-II-heme b(3) may weaken its affinity for NO and thus promotes, in the first catalytic step, binding of NO at the Fe-B(II) site. The function of heme b(3) would then be to orient the Fe-B-bound NO molecules for the formation of the N-N bond and to provide reducing equivalents for NO reduction.

}, author = {Timoteo, Cristina G. and Pereira, Alice S. and Martins, Carlos E. and Naik, Sunil G. and Duarte, Americo G. and Moura, Jose J. G. and Tavares, Pedro and Huynh, Boi Hanh and Moura, Isabel} } @article {5335, title = {New spectroscopic and electrochemical insights on a class I superoxide reductase: evidence for an intramolecular electron-transfer pathway}, journal = {BIOCHEMICAL JOURNAL}, volume = {{438}}, number = {{3}}, year = {2011}, note = {n/a}, pages = {{485-494}}, abstract = {

SORs (superoxide reductases) are enzymes involved in bacterial resistance to reactive oxygen species, catalysing the reduction of superoxide anions to hydrogen peroxide. So far three structural classes have been identified. Class I enzymes have two ironcentre-containing domains. Most studies have focused on the catalytic iron site (centre II), yet the role of centre I is poorly understood. The possible roles of this iron site were approached by an integrated study using both classical and fast kinetic measurements, as well as direct electrochemistry. A new heterometallic form of the protein with a zinc-substituted centre I, maintaining the iron active-site centre II, was obtained, resulting in a stable derivative useful for comparison with the native all-iron from. Second-order rate constants for the electron transfer between reduced rubredoxin and the different SOR forms were determined to be 2.8 x 10(7) M(-1) . s(-1) and 1.3 x 10(6) M(-1) . s(-1) for SOR(Fe(IIII)-Fe(II)) and for SOR(Fe(IIII)-Fe(III)) forms respectively, and 3.2 x 10(6) M(-1) s(-1) for the SOR(Zn(II)-Fe(III)) form. The results obtained seem to indicate that centre I transfers electrons from the putative physiological donor rubredoxin to the catalytic active iron site (intramolecular process). In addition, electrochemical results show that conformational changes are associated with the redox state of centre I, which may enable a faster catalytic response towards superoxide anion. The apparent rate constants calculated for the SOR-mediated electron transfer also support this observation.

}, author = {Folgosa, Filipe and Cordas, Cristina M. and Santos, Joana A. and Pereira, Alice S. and Moura, Jose J. G. and Tavares, Pedro and Moura, Isabel} } @article {5338, title = {Short communication: Effect of kefir grains on proteolysis of major milk proteins}, journal = {JOURNAL OF DAIRY SCIENCE}, volume = {{93}}, number = {{1}}, year = {2010}, note = {n/a}, month = {Feb}, pages = {{27-31}}, abstract = {

The effect of kefir grains on the proteolysis of major milk proteins in milk kefir and in a culture of kefir grains in pasteurized cheese whey was followed by reverse phase-HPLC analysis. The reduction of kappa-, alpha-, and beta-caseins (CN), alpha-lactalbumin (alpha-LA), and beta-lactoglobulin (beta-LG) contents during 48 and 90 h of incubation of pasteurized milk (100 mL) and respective cheese whey with kefir grains (6 and 12 g) at 20 degrees C was monitored. Significant proteolysis of alpha-LA and kappa-, alpha-, and beta-caseins was observed. The effect of kefir amount (6 and 12 g/100 mL) was significant for alpha-LA and alpha- and beta-CN. alpha-Lactalbumin and beta-CN were more easily hydrolyzed than alpha-CN. No significant reduction was observed with respect to beta-LG concentration for 6 and 12 g of kefir in 100 mL of milk over 48 h, indicating that no significant proteolysis was carried out. Similar results were observed when the experiment was conducted over 90 h. Regarding the cheese whey kefir samples, similar behavior was observed for the proteolysis of alpha-LA and beta-LG: alpha-LA was hydrolyzed between 60 and 90\% after 12 h (for 6 and 12 g of kefir) and no significant beta-LG proteolysis occurred. The proteolytic activity of lactic acid bacteria and yeasts in kefir community was evaluated. Kefir milk prepared under normal conditions contained peptides from proteolysis of alpha-LA and kappa-, alpha-, and beta-caseins. Hydrolysis is dependent on the kefir: milk ratio and incubation time. beta-Lactoglobulin is not hydrolyzed even when higher hydrolysis time is used. Kefir grains are not appropriate as adjunct cultures to increase beta-LG digestibility in whey-based or whey-containing foods.

}, author = {Ferreira, I M P L V O and Pinho, O and Monteiro, D and Faria, S and Cruz, S and Perreira, A and Roque, A C and Tavares, P} } @article {5339, title = {Isolation and characterization of a new Cu-Fe protein from Desulfovibrio aminophilus DSM12254}, journal = {Journal Of Inorganic Biochemistry}, volume = {{103}}, number = {{10, SI}}, year = {2009}, note = {

1st Latin American Meeting on Biological Inorganic Chemistry (LABIC2008), Iguazu Falls, BRAZIL, AUG 31-SEP 04, 2008

}, month = {Oct}, pages = {{1314-1322}}, abstract = {

The isolation and characterization of a new metalloprotein containing Cu and Fe atoms is reported. The as-isolated Cu-Fe protein shows an UV-visible spectrum with absorption bands at 320 nm, 409 nm and 615 nm. Molecular mass of the native protein along with denaturating electrophoresis and mass spectrometry data show that this protein is a multimer consisting of 14 +/- 1 subunits of 15254.3 +/- 7.6 Da. Mossbauer spectroscopy data of the as-isolated Cu-Fe protein is consistent with the presence of [2Fe-2S](2+) centers. Data interpretation of the dithionite reduced protein suggest that the metallic cluster could be constituted by two ferromagnetically coupled [2Fe-2S](+) spin delocalized pairs. The biochemical properties of the Cu-Fe protein are similar to the recently reported molybdenum resistance associated protein from Desulfovibrio, D. alaskensis. Further-more, a BLAST search from the DNA deduced amino acid sequence shows that the Cu-Fe protein has homology with proteins annotated as zinc resistance associated proteins from Desulfovibrio, D. alaskensis, D. vulgaris Hildenborough, D. piger ATCC 29098. These facts suggest a possible role of the Cu-Fe protein in metal tolerance. (C) 2009 Published by Elsevier Inc.

}, author = {Rivas, Maria G. and Mota, Cristiano S. and Pauleta, Sofia R. and Carepo, Marta S. P. and Folgosa, Filipe and Andrade, Susana L. A. and Fauque, Guy and Pereira, Alice S. and Tavares, Pedro and Calvete, Juan J. and Moura, Isabel and Moura, Jose J. G.} } @article {5340, title = {Camelid nanobodies raised against an integral membrane enzyme, nitric oxide reductase}, journal = {PROTEIN SCIENCE}, volume = {{18}}, number = {{3}}, year = {2009}, note = {n/a}, month = {Apr}, pages = {{619-628}}, abstract = {

Nitric Oxide Reductase (NOR) is an integral membrane protein performing the reduction of NO to N(2)O. NOR is composed of two subunits: the large one (NorB) is a bundle of 12 transmembrane helices (TMH). It contains a b type heme and a binuclear iron site, which is believed to be the catalytic site, comprising a heme b and a non-hemic iron. The small subunit (NorC) harbors a cytochrome c and is attached to the membrane through a unique TMH. With the aim to perform structural and functional studies of NOR, we have immunized dromedaries with NOR and produced several antibody fragments of the heavy chain (VHHs, also known as nanobodies (TM)). These fragments have been used to develop a faster NOR purification procedure, to proceed to crystallization assays and to analyze the electron transfer of electron donors. BIAcore experiments have revealed that up to three VHHs can bind concomitantly to NOR with affinities in the nanomolar range. This is the first example of the use of VHHs with an integral membrane protein. Our results indicate that VHHs are able to recognize with high affinity distinct epitopes on this class of proteins, and can be used as versatile and valuable tool for purification, functional study and crystallization of integral membrane proteins.

}, author = {Conrath, Katja and Pereira, Alice S. and Martins, Carlos E. and Timoteo, Cristina G. and Tavares, Pedro and Spinelli, Silvia and Kinne, Joerg and Flaudrops, Christophe and Cambillau, Christian and Muyldermans, Serge and Moura, Isabel and Moura, Jose J. G. and Tegoni, Mariella and Desmyter, Aline} } @article {5319, title = {Molybdenum Induces the Expression of a Protein Containing a New Heterometallic Mo-Fe Cluster in Desulfovibrio alaskensis}, journal = {Biochemistry}, volume = {{48}}, number = {{5}}, year = {2009}, note = {n/a}, pages = {{873-882}}, publisher = {AMER CHEMICAL SOC}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, abstract = {

The characterization of a novel Mo-Fe protein (MorP) associated with a system that responds to Mo in Desulfovibrio alaskensis is reported. Biochemical characterization shows that MorP is a periplasmic homomultimer of high molecular weight (260 +/- 13 kDa) consisting of 16-18 monomers of 15321.1 +/- 0.5 Da. The UV/visible absorption spectrum of the as-isolated protein shows absorption peaks around 280, 320, and 570 nm with extinction coefficients of 18700, 12800, and 5000 M(-1) cm(-1), respectively. Metal content, EXAFS data and DFT calculations support the presence of a Mo-2S-[2Fe-2S]-2S-Mo cluster never reported before. Analysis of the available genomes from Desulfovibrio species shows that the MorP encoding gene is located downstream of a sensor and a regulator gene. This type of gene arrangement, called two component system, is used by the cell to regulate diverse physiological processes in response to changes in environmemtal conditions. Increase of both gene expression and protein production was observed when cells were cultured in the presence of 45 mu M molybdenum. Involvement of this system in Mo tolerance of sulfate reducing bacteria is proposed.

}, author = {Rivas, Maria G. and Carepo, Marta S. P. and Mota, Cristiano S. and Korbas, Malgorzata and Durand, Marie-Claire and Lopes, Ana T. and Brondino, Carlos D. and Pereira, Alice S. and George, Graham N. and Dolla, Alain and Moura, Jose J. G. and Moura, Isabel} } @article {5320, title = {Electron transfer complex between nitrous oxide reductase and cytochrome c(552) from Pseudomonas nautica: Kinetic, nuclear magnetic resonance, and docking studies}, journal = {Biochemistry}, volume = {{47}}, number = {{41}}, year = {2008}, note = {n/a}, pages = {{10852-10862}}, publisher = {AMER CHEMICAL SOC}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, abstract = {

The multicopper enzyme nitrous oxide reductase (N2OR) catalyzes the final step of denitrification, the two-electron reduction of N2O to N-2. This enzyme is a functional homodimer containing two different multicopper sites: CuA and CuZ. CuA is a binuclear copper site that transfers electrons to the tetranuclear copper sulfide CuZ, the catalytic site. In this study, Pseudomonas nautica cytochrome C-552 was identified as the physiological electron donor. The kinetic data show differences when physiological and artificial electron donors are compared [cytochrome vs methylviologen (MV)]. In the presence of cytochrome c(552), the reaction rate is dependent on the ET reaction and independent of the N2O concentration. With MV, electron donation is faster than substrate reduction. From the study of cytochrome c(552) concentration dependence, we estimate the following kinetic parameters: K-mc512 = 50.2 +/- 9.0 mu M and V-maxc551 1.8 +/- 10.6 units/mg. The N2O concentration dependence indicates a K-mN2O of 14.0 +/- 2.9 mu M using MV as the electron donor. The pH effect on the kinetic parameters is different when MV or cytochrome c(552) is used as the electron donor (pK(a) = 6.6 or 8.3, respectively). The kinetic study also revealed the hydrophobic nature of the interaction, and direct electron transfer studies showed that CuA is the center that receives electrons from the physiological electron donor. The formation of the electron transfer complex was observed by H-1 NMR protein-protein titrations and was modeled with a molecular docking program (BiGGER). The proposed docked complexes corroborated the ET studies giving a large number of solutions in which cytochrome c(552) is placed near a hydrophobic patch located around the CuA center.

}, author = {Dell\'Acqua, Simone and Pauleta, Sofia R. and Monzani, Enrico and Pereira, Alice S. and Casella, Luigi and Moura, Jose J. G. and Moura, Isabel} } @article {5341, title = {A new type of metal-binding site in cobalt- and zinc-containing adenylate kinases isolated from sulfate-reducers Desulfovibrio gigas and Desulfovibrio desulfuricans ATCC 27774}, journal = {Journal Of Inorganic Biochemistry}, volume = {{102}}, number = {{5-6}}, year = {2008}, note = {

13th International Conference on Biological Inorganic Chemistry, Vienna, AUSTRIA, JUL 15-20, 2007

}, pages = {{1380-1395}}, abstract = {

Adenylate kinase (AK) mediates the reversible transfer of phosphate groups between the adenylate nucleotides and contributes to the maintenance of their constant cellular level, necessary for energy metabolism and nucleic acid synthesis. The AK were purified from crude extracts of two sulfate-reducing bacteria (SRB), Desulfovibrio (D.) gigas NCIB 9332 and Desulfovibrio desulfuricans ATCC 27774, and biochemically and spectroscopically characterised in the native and fully cobalt- or zinc-substituted forms. These are the first reported adenylate kinases that bind either zinc or cobalt and are related to the subgroup of metal-containing AK found, in most cases, in Gram-positive bacteria. The electronic absorption spectrum is consistent with tetrahedral coordinated cobalt, predominantly via sulfur ligands, and is supported by EPR. The involvement of three cysteines in cobalt or zinc coordination was confirmed by chemical methods. Extended X-ray absorption fine structure (EXAFS) indicate that cobalt or zinc are bound by three cysteine residues and one histidine in the metal-binding site of the {\textquoteleft}{\textquoteleft}LID\'\' domain. The sequence (129)Cys-X(5)-His-X(15)-Cys-X(2)-Cys of the AK from D. gigas is involved in metal coordination and represents a new type of binding motif that differs from other known zinc-binding sites of AK. Cobalt and zinc play a structural role in stabilizing the LID domain. (C) 2008 Elsevier Inc. All rights reserved.

}, author = {Gavel, Olga Yu and Bursakov, Sergey A. and Di Rocco, Giulia and Trincao, Jose and Pickering, Ingrid J. and George, Graham N. and Calvete, Juan J. and Shnyrov, Valery L. and Brondino, Carlos D. and Pereira, Alice S. and Lampreia, Jorge and Tavares, Pedro and Moura, Jose J. G. and Moura, Isabel} } @article {5342, title = {Conformations generated during turnover of the Azotobacter vinelandii nitrogenase MoFe protein and their relationship to physiological function}, journal = {Journal Of Inorganic Biochemistry}, volume = {{101}}, number = {{11-12}}, year = {2007}, note = {n/a}, month = {Nov}, pages = {{1649-1656}}, abstract = {

Various S = 3/2 EPR signals elicited from wild-type and variant Azotobacter vinelandii nitrogenase MoFe proteins appear to reflect different conformations assumed by the FeMo-cofactor with different protonation states. To determine whether these presumed changes in protonation and conformation reflect catalytic capacity, the responses (particularly to changes in electron flux) of the alpha H195Q, alpha H195N, and alpha Q191 K variant MoFe proteins (where His at position 195 in the alpha subunit is replaced by Gln/Asn or Gln at position alpha-191 by Lys), which have strikingly different substrate-reduction properties, were studied by stopped-flow or rapid-freeze techniques. Rapid-freeze EPR at low electron flux (at 3-fold molar excess of wild-type Fe protein) elicited two transient FeMo-cofactor-based EPR signals within 1 s of initiating turnover under N-2 with the alpha H195Q and alpha H195N variants, but not with the alpha Q191K variant. No EPR signals attributable to P cluster oxidation were observed for any of the variants under these conditions. Furthermore, during turnover at low electron flux with the wild-type, alpha H195Q or alpha H195N MoFe protein, the longer-time 430-nm absorbance increase, which likely reflects P cluster oxidation, was also not observed (by stopped-flow spectrophotometry); it did, however, occur for all three MoFe proteins under higher electron flux. No 430-nm absorbance increase occurred with the alpha Q191K variant, not even at higher electron flux. This putative lack of involvement of the P cluster in electron transfer at low electron flux was confirmed by rapid-freeze Fe-57 Mossbauer spectroscopy, which clearly showed FeMo-factor reduction without P cluster oxidation. Because the wild-type, alpha H195Q and alpha H195N MoFe proteins can bind N-2, but alpha Q195K cannot, these results suggest that P cluster oxidation occurs only under high electron flux as required for N-2 reduction. (C) 2007 Elsevier Inc. All rights reserved.

}, author = {Fisher, Karl and Lowe, David J. and Tavares, Pedro and Pereira, Alice S. and Huynh, Boi Hanh and Edmondson, Dale and Newton, William E.} } @article {5345, title = {Preparation of ingredients containing an ACE-inhibitory peptide by tryptic hydrolysis of whey protein concentrates}, journal = {INTERNATIONAL DAIRY JOURNAL}, volume = {{17}}, number = {{5}}, year = {2007}, note = {n/a}, month = {Jun}, pages = {{481-487}}, abstract = {

This study describes the characterisation of whey protein hydrolysates obtained from tryptic hydrolysis to assess their application as ingredients with angiotensin-converting-enzyme (ACE) inhibitory action. The levels of a-lactalbumin (alpha-la) and P-lactoglobulin (beta-lg) remaining after hydrolysis were quantified. Peptides were separated by RP-HPLC, and Ala-Leu-Pro-Met-His-Ile-Arg (ALPMHIR), the most potent beta-lg-derived ACE-inhibitory peptide was monitored. A correlation curve was established for the production of this peptide as a function of hydrolysis time. Heat-induced gelation of hydrolysates was studied by small-deformation rheology. The gelation times and the strength of the final gels were highly dependent on the degree of hydrolysis. Smaller peptides liberated by hydrolysis contributed to the inability of whey protein hydrolysates to gel. (c) 2006 Elsevier Ltd. All rights reserved.

}, author = {Ferreira, I M P L V O and Pinho, O and Mota, M. V. and Tavares, P and Pereira, A. and Goncalves, M. P. and Torres, D. and Rocha, C. and Teixeira, J. A.} } @article {5343, title = {NMR assignment of the apo-form of a Desulfovibrio gigas protein containing a novel Mo-Cu cluster}, journal = {Biomolecular Nmr Assignments}, volume = {{1}}, number = {{1}}, year = {2007}, note = {n/a}, month = {Jul}, pages = {{81-83}}, abstract = {

We report the 98\% assignment of the apo-form of an orange protein, containing a novel Mo-Cu cluster isolated from Desulfovibrio gigas. This protein presents a region where backbone amide protons exchange fast with bulk solvent becoming undetectable. These residues were assigned using C-13-detection experiments.

}, author = {Pauleta, Sofia R. and Duarte, Americo G. and Carepo, Marta S. and Pereira, Alice S. and Tavares, Pedro and Moura, Isabel and Moura, Jose J. G.} } @article {5344, title = {Superoxide reductases}, journal = {European Journal of Inorganic Chemistry}, number = {{18}}, year = {2007}, note = {n/a}, month = {Jul}, pages = {{2569-2581}}, abstract = {

Reactive oxygen species (ROS), when in excess, are among the most deleterious species an organism can deal with. The physiological effects of ROS include amino acid chain cleavage, DNA degradation and lipid oxidation, among others. They can be formed in the cytoplasm in a variety of ways, including autooxidation reactions (FMN- and FAD-containing enzymes) and Fenton reactions as a result of the cytoplasmatic pool of iron ions. The superoxide anion (021, despite its short half-life in solution, is particularly pernicious as it can form other reactive ROS (such as the strong oxidant peroxynitrite) or oxidize and/or reduce cellular components. For strict anaerobic or microaerophilic bacteria it is of particular importance to be able to dispose of ROS in a controlled manner, especially if these organisms are temporarily exposed to air. This review aims to describe the structural characteristics of superoxide reductases (SORs) and mechanistic aspects of biological superoxide anion reduction. SORs can be considered the main class of enzymes behind the oxygen detoxification pathway of anaerobic and microaerophilic bacteria. The geometry of the active site (three classes have been described), the possible electron donors in vivo and the current hypothesis for the catalytic mechanism will be discussed. Some phylogenetic considerations are presented, regarding the primary structure of SORs currently available in genome databases. ((c) Wiley-VCH Verlag GmbH \& Co. KGaA, 69451 Weinheim, Germany, 2007).

}, author = {Pereira, Alice S. and Tavares, Pedro and Folgosa, Filipe and Almeida, Rui M. and Moura, Isabel and Moura, Jose J. G.} } @article {5346, title = {Development and validation of an HPLC/UV method for quantification of bioactive peptides in fermented milks}, journal = {JOURNAL OF LIQUID CHROMATOGRAPHY \& RELATED TECHNOLOGIES}, volume = {{30}}, number = {{13-16}}, year = {2007}, note = {n/a}, pages = {{2139-2147}}, abstract = {

The simultaneous separation and quantification of two casein peptides (IPP, VPP) presenting potent inhibitory activity of angiotensin-converting-enzyme (ACE) and casein in fermented milks was developed. Gradient elution was carried out at a flow-rate of 1 mL/min, using a mixture of two solvents. Solvent A was 0.1\% TFA in water and solvent B was acetonitrile-water-trifluoracetic acid 95:5:0.1. The effluent was monitored by UV detector at 214 nm. Calibration curves were constructed in the interval of 0.01-1.0 mg/mL for VPP, 0.005-1.0 mg/mL for IPP, and 0.05-3.0 mg/mL for casein. R 2 invariably exceeded 0.999. The detection limits were 0.004 for VPP, 0.002 mg/mL for IPP, and 0.02 mg/mL for casein. Repeatability of the method was evaluated by six consecutive injections of two standard solutions containing VPP, IPP, and casein. The RSD values for concentration were all below 5.08\%. Recovery studies were carried out to determine the accuracy of the method. Recoveries ranged between 88 and 98.2\%. The methodology was applied, not only, for the monitorization of VPP, IPP, and casein in commercial fermented milks labeled as presenting anti hypertensive properties, but also, in milk with different degrees of fermentation by L Helveticus, and in other commercial functional fermented milks, such as, those presenting cholesterol lowering properties.

}, author = {Ferreira, Isabel M. P. L. V. O. and Eca, Rosario and Pinho, Ol{\'\i}via and Tavares, Pedro and Pereira, Alice and Roque, Ana Cecilia} } @article {5350, title = {Kinetics studies of the superoxide-mediated electron transfer reactions between rubredoxin-type proteins and superoxide reductases}, journal = {JOURNAL OF BIOLOGICAL INORGANIC CHEMISTRY}, volume = {{11}}, number = {{4}}, year = {2006}, note = {n/a}, month = {Jul}, pages = {{433-444}}, abstract = {

In this work we present a kinetic study of the superoxide-mediated electron transfer reactions between rubredoxin-type proteins and members of the three different classes of superoxide reductases (SORs). SORs from the sulfate-reducing bacteria Desulfovibrio vulgaris (Dv) and D. gigas (Dg) were chosen as prototypes of classes I and II, respectively, while SOR from the syphilis spyrochete Treponema pallidum (Tp) was representative of class III. Our results show evidence for different behaviors of SORs toward electron acceptance, with a trend to specificity for the electron donor and acceptor from the same organism. Comparison of the different k (app) values, 176.9 +/- 25.0 min(-1) in the case of the Tp/Tp electron transfer, 31.8 +/- 3.6 min(-1) for the Dg/Dg electron transfer, and 6.9 +/- 1.3 min(-1) for Dv/Dv, could suggest an adaptation of the superoxide-mediated electron transfer efficiency to various environmental conditions. We also demonstrate that, in Dg, another iron-sulfur protein, a desulforedoxin, is able to transfer electrons to SOR more efficiently than rubredoxin, with a k (app) value of 108.8 +/- 12.0 min(-1), and was then assigned as the potential physiological electron donor in this organism.

}, author = {Auchere, F. and Pauleta, S. R. and Tavares, P and Moura, I. and Moura, J. J. G.} } @article {5347, title = {Metalloenzymes of the denitrification pathway}, journal = {Journal Of Inorganic Biochemistry}, volume = {{100}}, number = {{12}}, year = {2006}, note = {n/a}, month = {Dec}, pages = {{2087-2100}}, abstract = {

Denitrification, or dissimilative nitrate reduction, is an anaerobic process used by some bacteria for energy generation. This process is important in many aspects, but its environmental implications have been given particular relevance. Nitrate accumulation and release of nitrous oxide in the atmosphere due to excess use of fertilizers in agriculture are examples of two environmental problems where denitrification plays a central role. The reduction of nitrate to nitrogen gas is accomplished by four different types of metalloenzymes in four simple steps: nitrate is reduced to nitrite, then to nitric oxide, followed by the reduction to nitrous oxide and by a final reduction to dinitrogen. In this manuscript we present a concise updated review of the bioinorganic aspects of denitrification. (c) 2006 Elsevier Inc. All rights reserved.

}, author = {Tavares, P and Pereira, A. S. and Moura, J. J. G. and Moura, I.} } @article {5348, title = {Nitric oxide reductase: Direct electrochemistry and electrocatalytic activity}, journal = {Chembiochem}, volume = {{7}}, number = {{12}}, year = {2006}, note = {n/a}, month = {Dec}, pages = {{1878-1881}}, abstract = {n/a}, author = {Cordas, Cristina M. and Pereira, Alice S. and Martins, Carlos E. and Timoteo, Cristina G. and Moura, Isabel and Moura, Jose J. G. and Tavares, Pedro} } @article {5349, title = {Overexpression and purification of Treponema pallidum rubredoxin; kinetic evidence for a superoxide-mediated electron transfer with the superoxide reductase neelaredoxin}, journal = {JOURNAL OF BIOLOGICAL INORGANIC CHEMISTRY}, volume = {{9}}, number = {{7}}, year = {2004}, note = {n/a}, month = {Oct}, pages = {{839-849}}, abstract = {

Superoxide reductases are a class of non-haem iron enzymes which catalyse the monovalent reduction of the superoxide anion O-2(-) into hydrogen peroxide and water. Treponema pallidum (Tp), the syphilis spirochete, expresses the gene for a superoxide reductase called neelaredoxin, having the iron protein rubredoxin as the putative electron donor necessary to complete the catalytic cycle. In this work, we present the first cloning, overexpression in Escherichia coli and purification of the Tp rubredoxin. Spectroscopic characterization of this 6 Da protein allowed us to calculate the molar absorption coefficient of the 490 nm feature of ferric iron, epsilon=6.9+/-0.4 mM(-1) cm(-1). Moreover, the midpoint potential of Tp rubredoxin, determined using a glassy carbon electrode, was -76+/-5 mV. Reduced rubredoxin can be efficiently reoxidized upon addition of Na2IrCl6-oxidized neelaredoxin, in agreement with a direct electron transfer between the two proteins, with a stoichiometry of the electron transfer reaction of one molecule of oxidized rubredoxin per one molecule of neelaredoxin. In addition, in presence of a steady-state concentration of superoxide anion, the physiological substrate of neelaredoxin, reoxidation of rubredoxin was also observed in presence of catalytic amounts of superoxide reductase, and the rate of rubredoxin reoxidation was shown to be proportional to the concentration of neelaredoxin, in agreement with a bimolecular reaction, with a calculated k(app)=180 min(-1). Interestingly, similar experiments performed with a rubredoxin from the sulfate-reducing bacteria Desulfovibrio vulgaris resulted in a much lower value of k(app)=4.5 min(-1). Altogether, these results demonstrated the existence for a superoxide-mediated electron transfer between rubredoxin and neelaredoxin and confirmed the physiological character of this electron transfer reaction.

}, author = {Auchere, F. and Sikkink, R. and Cordas, C. and Raleiras, P. and Tavares, P and Moura, I. and Moura, J. J. G.} } @article {5323, title = {Antagonists Mo and Cu in a heterometallic cluster present on a novel protein (orange protein) isolated from Desulfovibrio gigas}, journal = {Journal Of Inorganic Biochemistry}, volume = {{98}}, number = {{5}}, year = {2004}, note = {

11th International Conference on Biological Inorganic Chemistry, Cairns, AUSTRALIA, JUL 19-23, 2003

}, month = {Jun}, pages = {{833-840}}, publisher = {ELSEVIER SCIENCE INC}, address = {360 PARK AVE SOUTH, NEW YORK, NY 10010-1710 USA}, abstract = {

An orange-coloured protein (ORP) isolated from Desulfovibrio gigas, a sulphate reducer, has been previously shown by extended X-ray absorption fine structure (EXAFS) to contain a novel mixed-metal sulphide cluster of the type [S2MoS2CuS2MoS2] [J. Am. Chem. Soc. 122 (2000) 8321]. We report here the purification and the biochemical/spectroscopic characterisation of this novel protein. ORP is a soluble monomeric protein (11.8 kDa). The cluster is non-covalently bound to the polypeptide chain. The presence of a MoS42- moiety in the structure of the cofactor contributes with a quite characteristic UV-Vis spectra, exhibiting an orange colour, with intense absorption peaks at 480 and 338 nm. Pure ORP reveals an Abs(480)/Abs(338) ratio of 0.535. The gene sequence coding for ORP as well as the amino acid sequence was determined. The putative biological function of ORP is discussed. (C) 2003 Elsevier Inc. All rights reserved.

}, author = {Bursakov, S. A. and Gavel, O. Y. and Di Rocco, G. and Lampreia, J. and Calvete, J. and Pereira, A. S. and Moura, J. J. G. and Moura, I.} } @article {5322, title = {Structural basis for the mechanism of Ca2+ activation of the di-heme cytochrome c peroxidase from Pseudomonas nautica 617}, journal = {Structure}, volume = {{12}}, number = {{6}}, year = {2004}, note = {n/a}, month = {Jul}, pages = {{961-973}}, publisher = {CELL PRESS}, address = {1100 MASSACHUSETTS AVE, CAMBRIDGE, MA 02138 USA}, abstract = {

Cytochrome c peroxidase (CCP) catalyses the reduction of H2O2 to H2O, an important step in the cellular detoxification process. The crystal structure of the di-heme CCP from Pseudomonas nautica 617 was obtained in two different conformations in a redox state with the electron transfer heme reduced. Form IN, obtained at pH 4.0, does not contain Ca2+ and was refined at 2.2 Angstrom resolution. This inactive form presents a closed conformation where the peroxidatic heme adopts a six-ligand coordination, hindering the peroxidatic reaction from taking place. Form OUT is Ca2+ dependent and was crystallized at pH 5.3 and refined at 2.4 Angstrom resolution. This active form shows an open conformation, with release of the distal histidine (His71) ligand, providing peroxide access to the active site. This is the first time that the active and inactive states are reported for a di-heme peroxidase.

}, author = {Dias, J. M. and Alves, T. and Bonifacio, C. and Pereira, A. S. and Trincao, J. and Bourgeois, D. and Moura, I. and Rom{\~a}o, M.J.} } @article {5321, title = {Paracoccus pantotrophus pseudoazurin is an electron donor to cytochrome c peroxidase}, journal = {Biochemistry}, volume = {{43}}, number = {{35}}, year = {2004}, note = {n/a}, pages = {{11214-11225}}, publisher = {AMER CHEMICAL SOC}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, abstract = {

The gene for pseudoazurin was isolated from Paracoccus pantotrophus LMD 52.44 and expressed in a heterologous system with a yield of 54.3 mg of pure protein per liter of culture. The gene and protein were shown to be identical to those from P. pantotrophus LMD 82.5. The extinction coefficient of the protein was re-evaluated and was found to be 3.00 mM(-1) cm(-1) at 590 nm. It was confirmed that the oxidized protein is in a weak monomer/dimer equilibrium that is ionic- strength-dependent. The pseudoazurin was shown to be a highly active electron donor to cytochrome c peroxidase, and activity showed an ionic strength dependence consistent with an electrostatic interaction. The pseudoazurin has a very large dipole moment, the vector of which is positioned at the putative electron-transfer site, His81, and is conserved in this position across a wide range of blue copper proteins. Binding of the peroxidase to pseudoazurin causes perturbation of a set of NMR resonances associated with residues on the His81 face, including a ring of lysine residues. These lysines are associated with acidic residues just back from the rim, the resonances of which are also affected by binding to the peroxidase. We propose that these acidic residues moderate the electrostatic influence of the lysines and so ensure that specific charge interactions do not form across the interface with the peroxidase.

}, author = {Pauleta, S. R. and Guerlesquin, F. and Goodhew, C. F. and Devreese, B. and Van Beeumen, J. and Pereira, A. S. and Moura, I. and Pettigrew, G. W.} } @article {5352, title = {Ca2+ and the bacterial peroxidases: the cytochrome c peroxidase from Pseudomonas stutzeri}, journal = {JOURNAL OF BIOLOGICAL INORGANIC CHEMISTRY}, volume = {{8}}, number = {{1-2}}, year = {2003}, note = {n/a}, month = {Feb}, pages = {{29-37}}, abstract = {

The production of cytochrome c peroxidase (CCP) from Pseudomonas (Ps.) stutzeri (ATCC 11607) was optimized by adjusting the composition of the growth medium and aeration of the culture. The protein was isolated and characterized biochemically and spectroscopically in the oxidized and mixed valence forms. The activity of Ps. stutzeri CCP was studied using two different ferrocytochromes as electron donors: Ps. stutzeri cytochrome C-551 (the physiological electron donor) and horse heart cytochrome c. These electron donors interact differently with Ps. stutzeri CCP, exhibiting different ionic strength dependence. The CCP from Paracoccus (Pa.) denitrificans was proposed to have two different Ca2+ binding sites: one usually occupied (site I) and the other either empty or partially occupied in the oxidized enzyme (site II). The Ps. stutzeri enzyme was purified in a form with tightly bound Ca2+. The affinity for Ca2+ in the mixed valence enzyme is so high that Ca2+ returns to it from the EGTA which was added to empty the site in the oxidized enzyme. Molecular mass determination by ultracentrifugation and behavior on gel filtration chromatography have revealed that this CCP is isolated as an active dimer, in contrast to the Pa. denitrificans CCP which requires added Ca2+ for formation of the dimer and also for activation of the enzyme. This is consistent with the proposal that Ca2+ in the bacterial peroxidases influences the monomer/dimer equilibrium and the transition to the active form of the enzyme. Additional Ca2+ does affect both the kinetics of oxidation of horse heart cytochrome c (but not cytochrome C-551) and higher aggregation states of the enzyme. This suggests the presence of a superficial Ca2+ binding site of low affinity.

}, author = {Tim{\'o}teo, C G and Tavares, P and Goodhew, C. F. and Duarte, L. C. and Jumel, K. and Girio, F. M. F. and Harding, S. and Pettigrew, G. W. and Moura, I.} } @article {5351, title = {Formation of a stable cyano-bridged dinuclear iron cluster following oxidation of the superoxide reductases from Treponema pallidum and Desulfovibrio vulgaris with K3Fe(CN)(6)}, journal = {INORGANIC CHEMISTRY}, volume = {{42}}, number = {{4}}, year = {2003}, note = {n/a}, pages = {{938-940}}, abstract = {

Superoxide reductases catalyze the monovalent reduction of superoxide anion to hydrogen peroxide. Spectroscopic evidence for the formation of a dinuclear cyano-bridged adduct after K3Fe-(CN)(6) oxidation of the superoxide reductases neelaredoxin from Treponema pallidum and desulfoferrodoxin from Desulfovibrio vulgaris was reported. Oxidation with K3Fe(CN)(6) reveals a band in the near-IR with lambda(max) at 1020 nm, coupled with an increase of the iron content by almost 2-fold. Fourier transform infrared spectroscopy provided additional evidence with CN-stretching vibrations at 2095, 2025-2030, and 2047 cm(-1), assigned to a ferrocyanide adduct of the enzyme. Interestingly, the low-temperature electronic paramagnetic resonance (EPR) spectra of oxidized TpNIr reveal at least three different species indicating structural heterogeneity in the coordination environment of the active site Fe ion. Given the likely 6-coordinate geometry of the active site Fe3+ ion in the ferrocyanide adduct, we propose that the rhombic EPR species can serve as a model of a hexacoordinate form of the active site.

}, author = {Auchere, F. and Raleiras, P. and Benson, L. and Venyaminov, S. Y. and Tavares, P and Moura, J. J. G. and Moura, I. and Rusnak, F.} } @article {5353, title = {Stoichiometric production of hydrogen peroxide and parallel formation of ferric multimers through decay of the diferric-peroxo complex, the first detectable intermediate in ferritin mineralization}, journal = {Biochemistry}, volume = {{41}}, number = {{45}}, year = {2002}, note = {n/a}, pages = {{13435-13443}}, abstract = {

The catalytic step that initiates formation of the ferric oxy-hydroxide mineral core in the central cavity of H-type ferritin involves rapid oxidation of ferrous ion by molecular oxygen (ferroxidase reaction) at a binuclear site (ferroxidase site) found in each of the 24 subunits. Previous investigators have shown that the first detectable reaction intermediate of the ferroxidase reaction is a diferric-peroxo intermediate, F-peroxo, formed within 25 ms, which then leads to the release of H2O2 and formation of ferric mineral precursors. The stoichiometric relationship between F-peroxo, H2O2, and ferric mineral precursors, crucial to defining the reaction pathway and mechanism, has now been determined. To this end, a horseradish peroxidase-catalyzed spectrophotometric method was used as an assay for H2O2. By rapidly mixing apo M ferritin from frog, Fe2+, and O-2 and allowing the reaction to proceed for 70 ms when F-peroxo has reached its maximum accumulation, followed by spraying the reaction mixture into the H2O2 assay solution, we were able to quantitatively determine the amount of H2O2 produced during the decay of F-peroxo. The correlation between the amount of H2O2 released with the amount of F-peroxo accumulated at 70 ms determined by Mossbauer spectroscopy showed that F-peroxo decays into H2O2 with a stoichiometry of 1 F-peroxo:H2O2. When the decay of F-peroxo was monitored by rapid freeze-quench Mossbauer spectroscopy, multiple diferric mu-oxo/mu-hydroxo complexes and small polynuclear ferric clusters were found to form at rate constants identical to the decay rate of F-peroxo. This observed parallel formation of multiple products (H2O2, diferric complexes, and small polynuclear clusters) from the decay of a single precursor (F-peroxo) provides useful mechanistic insights into ferritin mineralization and demonstrates a flexible ferroxidase site.

}, author = {Jameson, GNL and Jin, W and Krebs, C. and Perreira, AS and Tavares, P and Liu, XF and Theil, EC and Huynh, B. H.} } @article {5356, title = {Calcium in bacterial peroxidases - Pseudomonas stutzeri cytochrome c peroxidase}, journal = {Journal Of Inorganic Biochemistry}, volume = {{86}}, number = {{1}}, year = {2001}, note = {n/a}, month = {Aug}, pages = {{456}}, abstract = {n/a}, author = {Tim{\'o}teo, C G and Tavares, P and Pettigrew, G. W. and Moura, I.} } @article {5324, title = {Cloning of a novel Mo-Cu containing protein from Desulfovibrio.gigas}, journal = {Journal Of Inorganic Biochemistry}, volume = {{86}}, number = {{1}}, year = {2001}, note = {n/a}, month = {Aug}, pages = {{202}}, publisher = {ELSEVIER SCIENCE INC}, address = {655 AVENUE OF THE AMERICAS, NEW YORK, NY 10010 USA}, abstract = {n/a}, author = {Di Rocco, G. and Pereira, A. S. and Bursakov, S. A. and Gavel, O. Y. and Rusnak, F. and Lampreia, J. and Moura, J. J. G. and Moura, I.} } @article {5354, title = {Developmen of an electrochemical biosensor for nitrite determination}, journal = {Journal Of Inorganic Biochemistry}, volume = {{86}}, number = {{1}}, year = {2001}, note = {n/a}, month = {Aug}, pages = {{121}}, abstract = {n/a}, author = {Almeida, G. and Tavares, P and Lampreia, J. and Moura, J. J. G. and Moura, I.} } @article {5355, title = {Nitrous oxide reductase (N2OR) from Pseudomonas nautica 617}, journal = {Journal Of Inorganic Biochemistry}, volume = {{86}}, number = {{1}}, year = {2001}, note = {n/a}, month = {Aug}, pages = {{165}}, abstract = {n/a}, author = {Cabrito, I. and Pereira, A. S. and Tavares, P and Besson, S. and Brondino, C. and Hoffman, B. and Brown, K. and Tegoni, M. and Cambillau, C. and Moura, J. J. G. and Moura, I.} } @article {5325, title = {Structure-function studies of cytochrome c peroxidase from ps. nautica}, journal = {Journal Of Inorganic Biochemistry}, volume = {{86}}, number = {{1}}, year = {2001}, note = {n/a}, month = {Aug}, pages = {{122}}, publisher = {ELSEVIER SCIENCE INC}, address = {655 AVENUE OF THE AMERICAS, NEW YORK, NY 10010 USA}, abstract = {n/a}, author = {Alves, T. and Besson, S. and Pereira, A. S. and Pettigrew, G. W. and Moura, J. J. G. and Moura, I.} } @article {5358, title = {Mossbauer characterization of the iron-sulfur clusters in Desulfovibrio vulgaris hydrogenase}, journal = {Journal Of The American Chemical Society}, volume = {{123}}, number = {{12}}, year = {2001}, note = {n/a}, pages = {{2771-2782}}, abstract = {

The periplasmic hydrogenase of Desulfovibrio vulgaris (Hildenbourough) is an all Fe-containing hydrogenase. It contains two ferredoxin type [4Fe-4S] clusters, termed the F clusters, and a catalytic H cluster. Recent X-ray crystallographic studies on two Fe hydrogenases revealed that the H cluster is composed of two sub-clusters, a [4Fe-4S] cluster ([4Fe-4S]H) and-a binuclear Fe cluster ([2Fe]H), bridged by a cysteine sulfur. The aerobically purified D. vulgaris hydrogenase is stable in air. It is inactive and requires reductive activation. Upon reduction, the enzyme becomes sensitive to O(2) indicating that the reductive activation process is irreversible. Previous EPR investigations showed that upon reoxidation (under argon) the H cluster exhibits a rhombic EPR signal that is not seen in the as-purified enzyme, suggesting a conformational change in association with the reductive activation. For the purpose of gaining more information on the electronic properties of this unique H cluster and to understand further the reductive activation process, variable-temperature and variable-field Mossbauer spectroscopy has been used to characterize the Fe-S clusters in D. vulgaris hydrogenase poised at different redox states generated during a reductive titration, and in the GO-reacted enzyme. The data were successfully decomposed into spectral components corresponding to the F and H clusters,and characteristic parameters describing the electronic and magnetic properties of the F and H clusters were obtained. Consistent with the X-ray crystallographic results, the spectra of the H cluster can be understood as originating from an exchange coupled [4Fe-4S] - [2Fe] system. In particular, detailed analysis of the data reveals that the reductive activation begins with reduction of the [4Fe-4S]H cluster from the 2+ to the If state, followed by transfer of the reducing equivalent from the [4Fe-4S]H subcluster to the binuclear [2Fe]H subcluster. The results also reveal that binding of exogenous CO to the H cluster affects significantly the exchange coupling between the [4Fe-4S]H and the [2Fe]H subclusters. Implication of such a CO binding effect is discussed.

}, author = {Pereira, A. S. and Tavares, P and Moura, I. and Moura, J. J. G. and Huynh, B. H.} } @article {5327, title = {Rational reprogramming of the R2 subunit of Escherichia coli ribonucleotide reductase into a self-hydroxylating monooxygenase}, journal = {Journal Of The American Chemical Society}, volume = {{123}}, number = {{29}}, year = {2001}, note = {n/a}, pages = {{7017-7030}}, publisher = {AMER CHEMICAL SOC}, address = {1155 16TH ST, NW, WASHINGTON, DC 20036 USA}, abstract = {

The outcome of O-2 activation at the diiron(II) cluster in the R2 subunit of Escherichia coli (class I) ribonucleotide reductase has been rationally altered from the normal tyrosyl radical (Y122)(1) production to self-hydroxylation of a phenylalanine side-chain by two amino acid substitutions that leave intact the (histidine)(2)-(carboxylate)(4) ligand set characteristic of the diiron-carboxylate family. Iron ligand Asp (D) 84 was replaced with Glu (E), the amino acid found in the cognate position of the structurally similar diiron-carboxylate protein, methane monooxygenase hydroxylase (MMOH). We previously showed that this substitution allows accumulation of a mu -1,2-peroxodiiron(III) intermediate,(2 3) which does not accumulate in the wild-type (wt) protein and is probably a structural homologue of intermediate P (H-peroxo) in O-2 activation by MMOH.(4) In addition, the near-surface residue Trp (W) 48 was replaced with Phe (F), blocking transfer of the {\textquoteleft}{\textquoteleft}extra\'\' electron that occurs in wt R2 during formation of the formally Fe(LII)Fe(IV) cluster X.(5-7) Decay of the mu1,2-peroxodiiron(III) complex in R2-W38F/D84E gives an initial brown product, which contains very little YI22(.) and which converts very slowly (t(1/2) similar to 7 h) upon incubation at 0 degreesC to an intensely purple final product. X-ray crystallographic analysis of the purple product indicates that F208 has undergone epsilon -hydroxylation and the resulting phenol has shifted significantly to become st ligand to Fe2 of the diiron cluster. Resonance Raman (RR) spectra of the purple product generated with O-16(2) or O-18(2) show appropriate isotopic sensitivity in bands assigned to O-phenyl and Fe-O-phenyl vibrational modes, confirming that the oxygen of the Fe(III)-phenolate species is derived from Or. Chemical analysis, experiments involving interception of the hydroxylating intermediate with exogenous reductant, and Mossbauer and EXAFS characterization of the brown and purple species establish that F208 hydroxylation occurs during decay of the peroxo complex and formation of the initial brown product. The slow transition to the purple Fe(LII)-phenolate species is ascribed to a ligand rearrangement in which mu -O2- is lost and the F208-derived phenolate coordinates. The reprogramming to F208 monooxygenase requires both amino acid substitutions, as very little epsilon -hydroxyphenylalanine is formed and pathways leading to Y122(.) formation predominate in both R2-D84E and R2-W48F(2-7).

}, author = {Baldwin, J and Voegtli, WC and Khidekel, N and Moenne-Loccoz, P and Krebs, C. and Pereira, A. S. and Ley, BA and Huynh, B. H. and Loehr, T. M. and Riggs-Gelasco, PJ and Rosenzweig, AC and Bollinger, JM} } @article {5357, title = {Substitution of murine ferrochelatase glutamate-287 with glutamine or alanine leads to porphyrin substrate-bound variants}, journal = {BIOCHEMICAL JOURNAL}, volume = {{356}}, number = {{1}}, year = {2001}, note = {n/a}, pages = {{217-222}}, abstract = {

Ferrochelatase (EC 4.99.1.1) is the terminal enzyme of the haem biosynthetic pathway and catalyses iron chelation into the protoporphyrin IX ring. Glutamate-287 (E287) of murine mature ferrochelatase is a conserved residue in all known sequences of ferrochelatase, is present at the active site of the enzyme, as inferred from the Bacillus subtilis ferrochelatase three-dimensional structure, and is critical for enzyme activity. Substitution of E287 with either glutamine (Q) or alanine (A) yielded variants with lower enzymic activity than that of the wild-type ferrochelatase and with different absorption spectra from the wild-type enzyme. In contrast to the wild-type enzyme, the absorption spectra of the variants indicate that these enzymes, as purified, contain protoporphyrin IX. Identification and quantification of the porphyrin bound to the E287-directed variants indicate that approx. 80\% of the total porphyrin corresponds to protoporphyrin IX. Significantly, rapid stopped-flow experiments of the E287A and E287Q Variants demonstrate that reaction with Zn2+ results in the formation of bound Zn-protoporphyrin IX, indicating that the endogenously bound protoporphyrin IX can be used as a substrate. Taken together, these findings suggest that the structural strain imposed by ferrochelatase on the porphyrin substrate as a critical step in the enzyme catalytic mechanism is also accomplished by the E287A and E287Q variants, but without the release of the product. Thus E287 in murine ferrochelatase appears to be critical For the catalytic process by controlling the release of the product.

}, author = {Franco, R. and Pereira, A. S. and Tavares, P and Mangravita, A. and Barber, M. J. and Moura, I. and Ferreira, G. C.} } @article {5326, title = {A novel type of catalytic copper cluster in nitrous oxide reductase}, journal = {Nature Structural Biology}, volume = {{7}}, number = {{3}}, year = {2000}, note = {n/a}, month = {Apr}, pages = {{191-195}}, publisher = {NATURE PUBLISHING GROUP}, address = {75 VARICK ST, 9TH FLR, NEW YORK, NY 10013-1917 USA}, abstract = {

Nitrous oxide (N(2)O) is a greenhouse gas, the third most significant contributor to global warming. As a key process for N(2)O elimination from the biosphere, N(2)O reductases catalyze the two-electron reduction of N(2)O to N(2). These 2 x 65 kDa copper enzymes are thought to contain a CuA electron entry site, similar to that of cytochrome c oxidase, and a CuZ catalytic center. The copper anomalous signal was used to solve the crystal structure of N(2)O reductase from Pseudomonas nautica by multiwavelength anomalous dispersion, to a resolution of 2.4 Angstrom. The structure reveals that the CuZ center belongs to a new type of metal cluster, in which four copper ions are liganded by seven histidine residues. N(2)O binds to this center via a single copper ion. The remaining copper ions might act as an electron reservoir, assuring a fast electron transfer and avoiding the formation of dead-end products.

}, author = {Brown, K. and Tegoni, M. and Prudencio, M. and Pereira, A. S. and Besson, S. and Moura, J. J. and Moura, I. and Cambillau, C.} } @article {5359, title = {Purification, characterization, and preliminary crystallographic study of copper-containing nitrous oxide reductase from Pseudomonas nautica 617}, journal = {Biochemistry}, volume = {{39}}, number = {{14}}, year = {2000}, note = {n/a}, pages = {{3899-3907}}, abstract = {

The aerobic purification of Pseudomonas nautica 617 nitrous oxide reductase yielded two forms of the enzyme exhibiting different chromatographic behaviors. The protein contains six copper atoms per monomer, arranged in two centers named CUA and Cut. Cut could be neither oxidized nor further reduced under our experimental conditions, and exhibits a 4-line EPR spectrum (g(x)= 2.015, A(x) = 1.5 mT, g(y) = 2.071, A(y) = 2 mT, g(z) = 2.138, A(z) = 7 mT) and a strong absorption at similar to 640 nm. Cu-A can be stabilized in a reduced EPR-silent state and in an oxidized state with a typical 7-line EPR spectrum (g(x) g(y) = 2.021, A(x) = A(y) = 0 T, g(z) =0.178, A(z) = 4 mT) and absorption bands at 480, 540, and similar to 800 nm. The difference between the two purified forms of nitrous oxide reductase is interpreted as a difference in the oxidation state of the CuA center. In form A, CUA is predominantly oxidized (S = 1/2, Cu1.5+-Cu1.5+), while in form B it is mostly in the one-electron reduced state (S = 0, Cu1+-Cu1+). In both forms, Cu-Z remains reduced (S = 1/2). Complete crystallographic data at 2.4 Angstrom indicate that Cu-A is a binuclear site (similar to the site found in cytochrome c oxidase) and Cu-Z is a novel tetracopper cluster [Brown, K., et ai. (2000) Nat. Struct. Biol. (in press)]. The complete amino acid sequence of the enzyme was determined and comparisons made with sequences of other nitrous oxide reductases, emphasizing the coordination of the centers. A 10.3 kDa peptide copurified with both forms of nitrous oxide reductase shows strong homology with proteins of the heat-shock GroES chaperonin family.

}, author = {Prudencio, M. and Pereira, A. S. and Tavares, P and Besson, S. and Cabrito, I. and Brown, K. and Samyn, B. and Devreese, B. and Van Beeumen, J. and Rusnak, F. and Fauque, G. and Moura, J. J. G. and Tegoni, M. and Cambillau, C. and Moura, I.} } @article {5360, title = {Redox potential measurements of the Mycobacterium tuberculosis heme protein KatG and the isoniazid-resistant enzyme KatG(S315T): Insights into isoniazid activation}, journal = {Biochemistry}, volume = {{39}}, number = {{37}}, year = {2000}, note = {n/a}, pages = {{11508-11513}}, abstract = {

Mycobacterium tuberculosis KatG is a multifunctional heme enzyme responsible for activation of the antibiotic isoniazid. A KatG(S315T) point mutation is found in \>50\% of isoniazid-resistant clinical isolates. Since isoniazid activation is thought to involve an oxidation reaction, the redox potential of KatG was determined using cyclic voltammetry, square wave voltammetry, and spectroelectrochemical titrations. Isoniazid activation may proceed via a cytochrome P450-like mechanism. Therefore, the possibility that substrate binding by KatG leads to an increase in the heme redox potential and the possibility that KatG(S315T) confers isoniazid resistance by altering the redox potential were examined. Effects of the heme spin state on the reduction potentials of KatG and KatG(S315T) were also determined. Assessment of the Fe3+/Fe2+ couple gave a midpoint potential of ca. -50 mV for both KatG and KatG(S315T). In contrast to cytochrome P450s, addition of substrate had no significant effect on either the KatG or KatG(S315T) redox potential. Conversion of the heme to a low-spin configuration resulted in a -150 to -200 mV shift of the KatG and KatG(S315T) redox potentials. These results suggest that isoniazid resistance conferred by KatG(S315T) is not mediated through changes in the heme redox potential. The redox potentials of isoniazid were also determined using cyclic and square wave voltammetry, and the results provide evidence that the ferric KatG and KatG(S315T) midpoint potentials are too low to promote isoniazid oxidation without formation of a high-valent enzyme intermediate such as compounds I and IT or oxyferrous KatG.

}, author = {Wengenack, N. L. and Lopes, H. and Kennedy, M. J. and Tavares, P and Pereira, A. S. and Moura, I. and Moura, J. J. G. and Rusnak, F.} } @book {5386, title = {ADVANCES IN INORGANIC CHEMISTRY, VOL 47}, year = {1999}, note = {n/a}, abstract = {n/a} } @article {5362, title = {Biochemical and spectroscopic characterization of overexpressed fuscoredoxin from Escherichia coli}, journal = {Biochemical And Biophysical Research Communications}, volume = {{260}}, number = {{1}}, year = {1999}, note = {n/a}, pages = {{209-215}}, abstract = {

Fuscoredoxin is a unique iron containing protein of yet unknown function originally discovered in the sulfate reducers of the genus Desulfovibrio. It contains two iron-sulfur clusters: a cubane [4Fe-4S] and a mixed oxo- and sulfide-bridged 4Fe cluster of unprecedented structure. The recent determination of the genomic sequence of Escherichia coli (E. coli) has revealed a homologue of fuscoredoxin in this facultative microbe. The presence of this gene in E. coli raises interesting questions regarding the function of fuscoredoxin and whether this gene represents a structural homologue of the better-characterized Desulfovibrio proteins. In order to explore the latter, an overexpression system for the E. coli fuscoredoxin gene was devised. The gene was cloned from genomic DNA by use of the polymerase chain reaction into the expression vector pT7-7 and overexpressed in E. coli BL21(DE3) cells. After two chromatographic steps a good yield of recombinant protein was obtained (approximately 4 mg of pure protein per liter of culture). The purified protein exhibits an optical spectrum characteristic of the homologue from D. desulfuricans, indicating that cofactor assembly was accomplished. Iron analysis indicated that the protein contains circa 8 iron atoms/molecule which were shown by EPR and Mossbauer spectroscopies to be present as two multinuclear clusters, albeit with slightly altered spectroscopic features. A comparison of the primary sequences of fuscoredoxins is presented and differences on cluster coordination modes are discussed on the light of the spectroscopic data. (C) 1999 Academic Press.

}, author = {Pereira, A. S. and Tavares, P and Krebs, C. and Huynh, B. H. and Rusnak, F. and Moura, I. and Moura, J. J. G.} } @article {5364, title = {Cloning and overexpression of E.Coli fuscoredoxin}, journal = {Journal Of Inorganic Biochemistry}, volume = {{74}}, number = {{1-4}}, year = {1999}, note = {n/a}, pages = {{260}}, abstract = {n/a}, author = {Pamplona, A. and Pereira, A. S. and Tavares, P and Moura, I. and Rusnak, F. and Moura, J. J. G.} } @article {5365, title = {Copper-containing nitrous oxide reductase from Pseudomonas nautica: spectroscopic and redox properties}, journal = {Journal Of Inorganic Biochemistry}, volume = {{74}}, number = {{1-4}}, year = {1999}, note = {n/a}, pages = {{267}}, abstract = {n/a}, author = {Prudencio, M. and Pereira, A. S. and Tavares, P and Besson, S. and Moura, I.} } @article {5361, title = {Purification and characterization of a tungsten-containing formate dehydrogenase from Desulfovibrio gigas}, journal = {Biochemistry}, volume = {{38}}, number = {{49}}, year = {1999}, note = {n/a}, pages = {{16366-16372}}, abstract = {

An air-stable formate dehydrogenase (FDH), an enzyme that catalyzes the oxidation of formate to carbon dioxide, was purified from the sulfate reducing organism Desulfovibrio gigas (D. gigas) NCIB 9332. D. gigas FDH is a heterodimeric protein [alpha (92 kDa) and beta (29 kDa) subunits] and contains 7 +/- 1 Fe/protein and 0.9 +/- 0.1 W/protein, Selenium was not detected. The UV/visible absorption spectrum of D, gigas FDH is typical of an iron-sulfur protein. Analysis of pterin nucleotides yielded a content of 1.3 +/- 0.1 guanine monophosphate/mol of enzyme, which suggests a tungsten coordination with two molybdopterin guanine dinucleotide cofactors. Both Mossbauer spectroscopy performed on D. gigas FDH grown in a medium enriched with Fe-57 and EPR studies performed in the native and fully reduced state of the protein confirmed the presence of two [4Fe-4S] clusters. Variable-temperature EPR studies showed the presence of two signals compatible with an atom in a d(1) configuration albeit with an unusual relaxation behavior as compared to the one generally observed for W(V) ions.

}, author = {Almendra, M. J. and Brondino, C. D. and Gavel, O. and Pereira, A. S. and Tavares, P and Bursakov, S. and Duarte, R. and Caldeira, J. and Moura, J. J. G. and Moura, I.} } @article {5367, title = {Reactions of nitric oxide with the reduced non-heme diiron center of the soluble methane monooxygenase hydroxylase}, journal = {Biochemistry}, volume = {{38}}, number = {{14}}, year = {1999}, note = {n/a}, pages = {{4504-4513}}, abstract = {

The soluble methane monooxygenase system from Methylococcus capsulatus (Bath) catalyzes the oxidation of methane to methanol and water utilizing dioxygen at a non-heme, carboxylate-bridged diiron center housed in the hydroxylase (H) component. To probe the nature of the reductive activation of dioxygen in this system, reactions of an analogous molecule, nitric oxide, with the diiron(II) form of the enzyme (H-red) Were investigated by both continuous and discontinuous kinetics methodologies using optical, EPR, and Mossbauer spectroscopy. Reaction of NO with H-red affords a dinitrosyl species, designated H-dinitrosyl, with optical spectra (lambda(max) = 450 and 620 nm) and Mossbauer parameters (delta = 0.72 mm/s, Delta E-Q = 1.55 mm/s) similar to those of synthetic dinitrosyl analogues and of the dinitrosyl adduct of the reduced ribonucleotide reductase R2 (RNR-R2) protein. The H-dinitrosyl species models features of the H-peroxo intermediate formed in the analogous dioxygen reaction. In the presence of protein B, H-dinitrosyl builds up with approximately the same rate constant as H-peroxo (similar to 26 s(-1)) at 4 degrees C. In the absence of protein B, the kinetics of H-dinitrosyl formation were best fit with a biphasic A --\> B --\> C model, indicating the presence of an intermediate species between H-red and H-dinitrosyl. This result contrasts with the reaction of H-red with dioxygen, in which the H-peroxo intermediate forms in measurable quantities only in the presence of protein B. These findings suggest that protein B may alter the positioning but not the availability of coordination sites on iron for exogenous ligand binding and reactivity.

}, author = {Coufal, DE and Tavares, P and Pereira, A. S. and Hyunh, BH and Lippard, SJ} } @article {5366, title = {Redox potential of the heme protein KatG from Mycobacterium tuberculosis}, journal = {Journal Of Inorganic Biochemistry}, volume = {{74}}, number = {{1-4}}, year = {1999}, note = {n/a}, pages = {{336}}, abstract = {n/a}, author = {Wengenack, N. and Lopes, H. and Kennedy, M. and Tavares, P and Pereira, A. S. and Moura, I. and Moura, J. J. G. and Rusnak, F.} } @inbook {5368, title = {Simple and complex iron-sulfur proteins in sulfate reducing bacteria}, volume = {{47}}, year = {1999}, note = {n/a}, pages = {{361-419}}, abstract = {n/a}, author = {Moura, I. and Pereira, A. S. and Tavares, P and Moura, J. J. G.} } @article {5363, title = {Spectroscopic characterization of porphyrin binding to ferrochelatase, the last enzyme in the heme biosynthetic pathway}, journal = {Journal Of Inorganic Biochemistry}, volume = {{74}}, number = {{1-4}}, year = {1999}, note = {n/a}, pages = {{130}}, abstract = {n/a}, author = {Franco, R. and Ma, J. G. and Lu, Y. and Pereira, A. and Tavares, P and Moura, I. and Shelnutt, J. A. and Ferreira, G. C.} } @article {5369, title = {Direct spectroscopic and kinetic evidence for the involvement of a peroxodiferric intermediate during the ferroxidase reaction in fast ferritin mineralization}, journal = {Biochemistry}, volume = {{37}}, number = {{28}}, year = {1998}, note = {n/a}, pages = {{9871-9876}}, abstract = {

Rapid freeze-quench (RFQ) Mossbauer and stopped-flow absorption spectroscopy were used to monitor the ferritin ferroxidase reaction using recombinant (apo) frog M ferritin; the initial transient ferric species could be trapped by the RFQ method using low iron loading (36 Fe2+/ferritin molecule). Biphasic kinetics of ferroxidation were observed and measured directly by the Mossbauer method; a majority (85\%) of the ferrous ions was oxidized at a fast rate of similar to 80 s(-1) and the remainder at a much slower rate of similar to 1.7 s(-1). In parallel with the fast phase oxidation of the Fe2+ ions, a single transient iron species is formed which exhibits magnetic properties (diamagnetic ground state) and Mossbauer parameters (Delta E-Q = 1.08 +/- 0.03 mm/s and delta = 0.62 +/- 0.02 mm/s) indicative of an antiferromagnetically coupled peroxodiferric complex. The formation and decay rates of this transient diiron species measured by the RFQ Mossbauer method match those of a transient blue species (lambda(max) = 650 nm) determined by the stopped-flow absorbance measurement. Thus, the transient colored species is assigned to the same peroxodiferric intermediate. Similar transient colored species have been detected by other investigators in several other fast ferritins (H and M subunit types), such as the human H ferritin and the Escherichia coli ferritin, suggesting a similar mechanism for the ferritin ferroxidase step in all fast ferritins. Peroxodiferric complexes are also formed as early intermediates in the reaction of O-2 With the catalytic diiron centers in the hydroxylase component of soluble methane monooxygenase (MMOH) and in the D84E mutant of the R2 subunit of E. coli ribonucleotide reductase. The proposal that a single protein site, with a structure homologous to the diiron centers in MMOH and R2, is involved in the ferritin ferroxidation step is confirmed by the observed kinetics, spectroscopic properties, and purity of the initial peroxodiferric species formed in the frog M ferritin.

}, author = {Pereira, A. S. and Small, W and Krebs, C. and Tavares, P and Edmondson, D. E. and Theil, EC and Huynh, B. H.} } @article {5371, title = {Generation of a mixed-valent Fe(III)Fe(IV) form of intermediate Q in the reaction cycle of soluble methane monooxygenase, an analog of intermediate X in ribonucleotide reductase R2 assembly}, journal = {Journal Of The American Chemical Society}, volume = {{120}}, number = {{9}}, year = {1998}, note = {n/a}, pages = {{2190-2191}}, abstract = {n/a}, author = {Valentine, AM and Tavares, P and Pereira, A. S. and Davydov, R and Krebs, C. and Koffman, BM and Edmondson, D. E. and Huynh, B. H. and Lippard, SJ} } @article {5370, title = {Probing the iron environment in desulforedoxin. EXAFS of oxidized and reduced states}, journal = {INORGANICA CHIMICA ACTA}, volume = {{273}}, number = {{1-2}}, year = {1998}, note = {

33rd International Conference on Coordination Chemistry, FLORENCE, ITALY, AUG 30-SEP 04, 1998

}, pages = {{409-411}}, abstract = {

Fe XAS data were collected on the oxidized and reduced forms of desulforedoxin from Desulfovibrio gigas, the oxidized form of rubredoxin from Clostridium pasteurianum, and the reduced form of rubredoxin from Pyrococcus furiosus. Analysis of these data is consistent with tetrahedral FeS(4) coordination in both oxidation states, and an expansion of the Fe-S distances from 2.27 to 2.33 Angstrom upon reduction. (C) 1998 Elsevier Science S.A. All rights reserved.

}, author = {Stalhandske, CMV and Dong, J. and Tavares, P and Liu, M. Y. and Legall, J. and Moura, J. J. G. and Moura, I. and Park, J. B. and Adams, M. W. W. and Scott, R. A.} } @article {5372, title = {Spectroscopic characterization of a novel tetranuclear Fe cluster in an iron-sulfur protein isolated from Desulfovibrio desulfuricans}, journal = {Biochemistry}, volume = {{37}}, number = {{9}}, year = {1998}, note = {n/a}, pages = {{2830-2842}}, abstract = {

Mossbauer and EPR spectroscopies were used to characterize the Fe clusters in an Fe-S protein isolated from Desulfovibrio desulfuricans (ATCC 27774). This protein was previously thought to contain hexanuclear Fe clusters, but a recent X-ray crystallographic measurement on a similar protein isolated from Desulfovibrio vulgaris showed that the protein contains two tetranuclear clusters, a cubane-type [4Fe-4S] cluster and a mixed-ligand cluster of novel structure [Lindley et al. (1997) Abstract, Chemistry of Metals in Biological Systems, European Research Conference, Tomar, Portugal]. Three protein samples poised at different redox potentials (as-purified, 40 and 320 mV) were investigated. In all three samples, the [4Fe-4S] cluster was found to be present in the diamagnetic 2+ oxidation state and exhibited typical Mossbauer spectra. The novel-structure cluster was found to be redox active. In the 320-mV and as-purified samples, the cluster is at a redox equilibrium between its fully oxidized and one-electron reduced states. In the 40-mV sample, the cluster is in a two-electron reduced state. Distinct spectral components associated with the four Fe sites of cluster 2 in the three oxidation states were identified. The spectroscopic parameters obtained for the Fe sites reflect different ligand environments, making it possible to assign the spectral components to individual Fe sites. In the fully oxidized state, all four iron ions are high-spin ferric and antiferromagnetically coupled to form a diamagnetic S = 0 state. In the one-electron and two-electron reduced states, the reducing electrons were found to localize, consecutively, onto two Fe sites that are rich in oxygen/nitrogen ligands. Based on the X-ray structure and the Mossbauer parameters, attempts could be made to identify the reduced Fe sites. For the two-electron reduced cluster, EPR and Mossbauer data indicate that the cluster is paramagnetic with a nonzero interger spin. For the one-electron reduced cluster, the data suggest a half-integer spin of 9/2 Characteristic fine and hyperfine parameters for all four Fe sites were obtained. Structural implications and the nature of the spin-coupling interactions are discussed.

}, author = {Tavares, P and Pereira, A. S. and Krebs, C. and Ravi, N. and Moura, J. J. G. and Moura, I. and Huynh, B. H.} } @article {5376, title = {Conversion of desulforedoxin into a rubredoxin center}, journal = {Biochemical And Biophysical Research Communications}, volume = {{231}}, number = {{3}}, year = {1997}, note = {n/a}, pages = {{679-682}}, abstract = {

Rubredoxin and desulforedoxin both contain an Fe(S-Cys)(4) center, However the spectroscopic properties of the center in desulforedoxin differ from rubredoxin, These differences arise from a distortion of the metal site hypothesized to result from adjacent cysteine residues in the primary sequence of desulforedoxin. Two desulforedoxin mutants were generated in which either a G or P-V were inserted between adjacent cysteines. Both mutants exhibited optical spectra with maxima at 278, 345, 380, 480, and 560 nm while the low temperature X-band EPR spectra indicated high-spin Fe3+ ions with large rhombic distortions (E/D = 0.21-0.23). These spectroscopic properties are distinct from wild type desulforedoxin and virtually identical to rubredoxin. (C) 1997 Academic Press.

}, author = {Yu, L. and Kennedy, M. and Czaja, C. and Tavares, P and Moura, J. J. G. and Moura, I. and Rusnak, F.} } @article {5374, title = {Mossbauer spectroscopic and kinetic characterization of ferric clusters formed in h-chain ferritin mineralization.}, journal = {Abstracts Of Papers Of The American Chemical Society}, volume = {{213}}, number = {{2}}, year = {1997}, note = {n/a}, pages = {{503-INOR}}, abstract = {n/a}, author = {Tavares, P and Pereira, A. S. and Lloyd, S. G. and Danger, D and Edmondson, D. E. and Theil, EC and Huynh, B. H.} } @article {5373, title = {Rapid and parallel formation of Fe3+ multimers, including a trimer, during H-type subunit ferritin mineralization}, journal = {Biochemistry}, volume = {{36}}, number = {{25}}, year = {1997}, note = {n/a}, pages = {{7917-7927}}, abstract = {

Conversion of Fe ions in solution to the solid phase in ferritin concentrates iron required for cell function. The rate of the Fe phase transition in ferritin is tissue specific and reflects the differential expression of two classes of ferritin subunits (H and L). Early stages of mineralization were probed by rapid freeze-quench Mossbauer, at strong fields (up to 8 T), and EPR spectroscopy in an H-type subunit, recombinant frog ferritin; small numbers of Fe (36 moles/mol of protein) were used to increase Fe3+ in mineral precursor forms, At 25 ms, four Fe3+-oxy species (three Fe dimers and one Fe trimer) were identified, These Fe3+-oxy species were found to form at similar rates and decay subsequently to a distinctive superparamagentic species designated the {\textquoteleft}\'young core.\'\' The rate of oxidation of Fe2+ (1026 s(-1)) corresponded well to the formation constant for the Fe3+- tyrosinate complex (920 s(-1)) observed previously [Waldo, G. S., \& Theil, E. C. (1993) Biochemistry 32, 13261] and, coupled with EPR data, indicates that several or possibly all of the Fe3+-oxy species involve tyrosine. The results, combined with previous Mossbauer studies of Y30F human H-type ferritin which showed decreases in several Fe3+ intermediates and stabilization of Fe2+ [Bauminger, E. R., et al. (1993) Biochem, J. 296, 709], emphasize the involvement of tyrosyl residues in the mineralization of H-type ferritins. The subsequent decay of these multiple Fe3+-oxy species to the superparamagnetic mineral suggests that Fe3+ species in different environments may be translocated as intact units from the protein shell into the ferritin cavity where the conversion to a solid mineral occurs.

}, author = {Pereira, A. S. and Tavares, P and Lloyd, S. G. and Danger, D and Edmondson, D. E. and Theil, EC and Huynh, B. H.} } @article {5377, title = {Preliminary crystallographic analysis of the oxidized form of a two mono-nuclear iron centres protein from Desulfovibrio desulfuricans ATCC 27774}, journal = {PROTEIN SCIENCE}, volume = {{5}}, number = {{6}}, year = {1996}, note = {n/a}, month = {Jul}, pages = {{1189-1191}}, abstract = {

Crystals of the fully oxidized form of desulfoferrodoxin were obtained by vapor diffusion from a solution containing 20\% PEG 4000, 0.1 M HEPES buffer, pH 7.5, and 0.2 M CaCl2. Trigonal and/or rectangular prisms could be obtained, depending on the temperature used for the crystal growth. Trigonal prisms belong to the rhombohedral space group R32, with a = 112.5 Angstrom and c = 63.2 Angstrom; rectangular prisms belong to the monoclinic space group C2, with a = 77.7 Angstrom, b = 80.9 Angstrom, c = 53.9 Angstrom, and beta = 98.1 degrees. The crystallographic asymmetric unit of the rhombohedral crystal form contains one molecule. There are two molecules in the asymmetric unit of the monoclinic form, in agreement with the self-rotation function.

}, author = {Coelho, A. V. and Matias, P. M. and Carrondo, M. A. and Tavares, P and Moura, J. J. G. and Moura, I. and Fulop, V. and Hajdu, J. and Legall, J.} } @article {5375, title = {The solution structure of desulforedoxin, a simple iron-sulfur protein - An NMR study of the zinc derivative}, journal = {JOURNAL OF BIOLOGICAL INORGANIC CHEMISTRY}, volume = {{1}}, number = {{4}}, year = {1996}, note = {n/a}, month = {Aug}, pages = {{341-354}}, abstract = {

Desulforedoxin is a simple dimeric protein isolated from Desulfovibrio gigas containing a distorted rubredoxin-like center with one iron coordinated by four cysteinyl residues (7.9 kDa with a 36-amino-acid monomer). H-1 NMR spectra of the oxidized Dx(Fe3+) and reduced Dx(Fe2+) forms were analyzed. The spectra show substantial line broadening due to the paramagnetism of iron. However, very low-field-shifted resonances, assigned to H beta protons, were observed in the reduced state and their temperature dependence analyzed. The active site of Dx was reconstituted with zinc, and its solution structure was determined using 2D NMR methods. This diamagnetic form gave high-resolution NMR data enabling the identification of all the amino acid spin systems. Sequential assignment and the determination of secondary structural elements was attempted using 2D NOESY experiments. However, because of the symmetrical dimer nature of the protein standard, NMR sequential assignment methods could not resolve all cross peaks due to inter- and intra-chain effects. The X-ray structure enabled the spatial relationship between the monomers to be obtained, and resolved the assignment problems. Secondary structural features could be identified from the NMR data; an antiparallel beta-sheet running from D5 to V18 with a well-defined beta-turn around cysteines C9 and C12. The section G22 to T25 is poorly defined by the NMR data and is followed by a turn around V27-C29. The C-terminus ends up near residues V6 and Y7. Distance geometry (DG) calculations allowed families of structures to be generated from the NMR data. A family of structures with a low target function violation for the Dr monomer and dimer were found to have secondary structural elements identical to those seen in the X-ray structure. The amide protons for G4, D5, G13, L11 NH and Q14 NH epsilon amide protons, H-bonded in the X-ray structure, were not seen by NMR as slowly exchanging, while structural disorder at the N-terminus, for the backbone at E10 and for the section G22-T25, was observed. Comparison between the Fe and Zn forms of Dr suggests that metal substitution does not have an effect on the structure of the protein.

}, author = {Goodfellow, B. J. and Tavares, P and Rom{\~a}o, M.J. and Czaja, C. and Rusnak, F. and Legall, J. and Moura, I. and Moura, J. J. G.} } @article {5328, title = {Characterization of representative enzymes from a sulfate reducing bacterium implicated in the corrosion of steel}, journal = {Biochemical And Biophysical Research Communications}, volume = {{221}}, number = {{2}}, year = {1996}, note = {n/a}, pages = {{414-421}}, publisher = {ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS}, address = {525 B ST, STE 1900, SAN DIEGO, CA 92101-4495}, abstract = {

This communication reports the isolation, purification and characterization of key enzymes involved in dissimilatory sulfate reduction of a sulfate reducing bacterium classified as Desulfovibrio desulfuricans subspecies desulfuricans New Jersey (NCIMB 8313) (Ddd NJ). The chosen strain, originally recovered from a corroding cast iron heat exchanger, was grown in large scale batch cultures. Physico-chemical and spectroscopic studies of the purified enzymes were carried out. These analyses revealed a high degree of similarity between proteins isolated from the DddNJ strain and the homologous proteins obtained from Desulfomicrobium baculatus Norway 4. In view of the results obtained, taxonomic reclassification of Desulfovibrio desulfuricans subspecies desulfuricans New Jersey (NCIMB 8313) into Desulfomicrobium baculatus (New Jersey) is proposed. (C) 1996 Academic Press, Inc.

}, author = {Pereira, A. S. and Franco, R. and Feio, M. J. and Pinto, C. and Lampreia, J. and Reis, M. A. and Calvete, J. and Moura, I. and Beech, I. and Lino, A. R. and Moura, J. J. G.} } @article {5378, title = {Primary structure of desulfoferrodoxin from Desulfovibrio desulfuricans ATCC 27774, a new class of non-heme iron proteins}, journal = {FEBS Letters}, volume = {{385}}, number = {{3}}, year = {1996}, note = {n/a}, pages = {{138-142}}, abstract = {

The primary structure of desulfoferrodoxin from Desulfovibrio desulfuricans ATCC 27774, a redox protein with two mononuclear iron sites, was determined by automatic Edman degradation and mass spectrometry of the composing peptides, It contains 125 amino acid residues of which five are cysteines, The first four, Cys-9, Cys-12, Cys-28 and Cys-29, are responsible for the binding of Center I which has a distorted tetrahedral sulfur coordination similar to that found in desulforedoxin from D. gigas, The remaining Cys-115 is proposed to be involved in the coordination of Center II, which is probably octahedrally coordinated with predominantly nitrogen/oxygen containing ligands as previously suggested by Mossbauer and Raman spectroscopy.

}, author = {Devreese, B. and Tavares, P and Lampreia, J. and Van Damme, N. and Legall, J. and Moura, J. J. G. and Van Beeumen, J. and Moura, I.} } @article {5379, title = {CRYSTAL-STRUCTURE OF DESULFOREDOXIN FROM DESULFOVIBRIO-GIGAS DETERMINED AT 1.8 ANGSTROM RESOLUTION - A NOVEL NONHEME IRON PROTEIN-STRUCTURE}, journal = {JOURNAL OF MOLECULAR BIOLOGY}, volume = {{251}}, number = {{5}}, year = {1995}, note = {n/a}, pages = {{690-702}}, abstract = {

The crystal structure of desulforedoxin from Desulfovibrio gigas, a new homo-dimeric (2x36 amino acids) non-heme iron protein, has been solved by the SIRAS method using the indium-substituted protein as the single derivative. The structure was refined to a crystallographic X-factor of 16.9\% at 1.8 Angstrom resolution. Native desulforedoxin crystals were grown from either PEG 4K or lithium sulfate, with cell constants a = b = 42.18 Angstrom, = 72.22 Angstrom (for crystals grown from PEG 4K), and they belong to space group P3(2)21. The indium-substituted protein crystallized isomorphously under the same conditions. The 2-fold symmetric dimer is firmly hydrogen bonded and folds as an incomplete beta-barrel with the two iron centers placed on opposite poles of the molecule. Each iron atom is coordinated to four cysteinyl residues in a distorted tetrahedral arrangement. Both iron atoms are 16 Angstrom apart but connected across the 2-fold axis by 14 covalent bonds along the polypeptide chain plus two hydrogen bonds. Desulforedoxin and rubredoxin share some structural features but show significant differences in terms of metal environment and water structure, which account for the known spectroscopic differences between rubredoxin and desulforedoxin. (C) 1995 Academic Press Limited

}, author = {Archer, M. and Huber, R. and Tavares, P and Moura, I. and Moura, J. J. G. and Carrondo, M. A. and Sieker, L. C. and Legall, J. and Rom{\~a}o, M.J.} } @article {5380, title = {EXPRESSION OF DESULFOVIBRIO-GIGAS DESULFOREDOXIN IN ESCHERICHIA-COLI - PURIFICATION AND CHARACTERIZATION OF MIXED-METAL ISOFORMS}, journal = {Journal Of Biological Chemistry}, volume = {{270}}, number = {{35}}, year = {1995}, note = {n/a}, pages = {{20273-20277}}, abstract = {

The dsr gene from Desulfovibrio gigas encoding the nonheme iron protein desulforedoxin was cloned using the polymerase chain reaction, expressed in Escherichia coli, and purified to homogeneity. The physical and spectroscopic properties of the recombinant protein resemble those observed for the native protein isolated from D. gigas. These include an alpha(2) tertiary structure, the presence of bound iron, and absorbance maxima at 370 and 506 nm in the UV/visible spectrum due to ligand-to-iron charge transfer bands. Low temperature electron paramagnetic resonance studies confirm the presence of a high spin ferric ion with g values of 7.7, 5.7, 4.1, and 1.8. Interestingly, E. coli produced two forms of desulforedoxin containing iron. One form was identified as a dimer with the metal-binding sites of both subunits occupied by iron while the second form contained equivalent amounts of iron and zinc and represents a dimer with one subunit occupied by iron and the second with zinc.

}, author = {Czaja, C. and Litwiller, R. and Tomlinson, A. J. and Naylor, S. and Tavares, P and Legall, J. and Moura, J. J. G. and Moura, I. and Rusnak, F.} } @article {5381, title = {TOTAL SYNTHESIS OF A SIMPLE METALLOPROTEIN - DESULFOREDOXIN}, journal = {Biochemical And Biophysical Research Communications}, volume = {{208}}, number = {{2}}, year = {1995}, note = {n/a}, pages = {{680-687}}, abstract = {

Desulforedoxin is a protein purified from cellular extracts of Desulfovibrio gigas. It is a small (7.9 kDa) dimeric protein that contains a distorted rubredoxin like center (one single iron coordinated by four cysteinyl residues). Due to the simplicity of the polypeptide chain and of the iron center, an attempt was made to chemically produce this protein. A 36 amino acid polypeptide chain was synthesized based on the known sequence of native Desulforedoxin. The iron center was then reconstituted and the biochemical and spectroscopic characteristics of this synthetic protein were investigated. The final product has an equal sequence to the protein purified from D. gigas. The synthetic and natural Dr are very similar, in terms of redox potential and spectroscopic properties (UV-Visible, EPR, Mossbauer). (C) 1995 Academic Press, Inc.

}, author = {Tavares, P and Wunderlich, J. K. and Lloyd, S. G. and Legall, J. and Moura, J. J. G. and Moura, I.} } @inbook {5330, title = {ADENYLYLSULFATE REDUCTASES FROM SULFATE-REDUCING BACTERIA}, volume = {{243}}, year = {1994}, note = {n/a}, pages = {{241-260}}, publisher = {ACADEMIC PRESS INC}, organization = {ACADEMIC PRESS INC}, address = {525 B STREET, SUITE 1900, SAN DIEGO, CA 92101-4495}, abstract = {n/a}, author = {Lampreia, J. and Pereira, A. S. and Moura, J. J. G.} } @inbook {5383, title = {CHARACTERIZATION OF 3 PROTEINS CONTAINING MULTIPLE IRON SITES - RUBRERYTHRIN, DESULFOFERRODOXIN, AND A PROTEIN CONTAINING A 6-IRON CLUSTER}, volume = {{243}}, year = {1994}, note = {n/a}, pages = {{216-240}}, abstract = {n/a}, author = {Moura, I. and Tavares, P and Ravi, N.} } @book {5331, title = {INORGANIC MICROBIAL SULFUR METABOLISM}, year = {1994}, note = {n/a}, publisher = {ACADEMIC PRESS INC}, organization = {ACADEMIC PRESS INC}, address = {525 B STREET, SUITE 1900, SAN DIEGO, CA 92101-4495}, abstract = {n/a} } @article {5329, title = {MAMMALIAN FERROCHELATASE, A NEW ADDITION TO THE METALLOENZYME FAMILY}, journal = {Journal Of Biological Chemistry}, volume = {{269}}, number = {{10}}, year = {1994}, note = {n/a}, pages = {{7062-7065}}, publisher = {AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC}, address = {9650 ROCKVILLE PIKE, BETHESDA, MD 20814}, abstract = {

A [2Fe-2S] cluster has been detected in mammalian ferrochelatase, the terminal enzyme of the heme biosynthetic pathway. Natural ferrochelatase, purified from mouse livers, and recombinant ferrochelatase, purified from an overproducing strain of Escherichia coli, were investigated by electron paramagnetic resonance (EPR) and Mossbauer spectroscopy. In their reduced forms, both the natural and recombinant ferrochelatases exhibited an identical EPR signal with g values (g = 2.00, 1.93, and 1.90) and relaxation properties typical of [2Fe-2S](+) cluster. Mossbauer spectra of the recombinant ferrochelatase, purified from a strain of E. coli cells transformed with a plasmid encoding murine liver ferrochelatase and grown in Fe-57-enriched medium, demonstrated unambiguously that the cluster is a [2Fe-2S] cluster. No change in the cluster oxidation state was observed during catalysis, The putative protein binding site for the Fe-S cluster in mammalian ferrochelatases is absent from the sequences of the bacterial and yeast enzymes, suggesting a possible role of the [2Fe-2S] center in regulation of mammalian ferrochelatases.

}, author = {Ferreira, G. C. and Franco, R. and Lloyd, S. G. and Pereira, A. S. and Moura, I. and Moura, J. J. G. and Huynh, B. H.} } @article {5382, title = {SPECTROSCOPIC PROPERTIES OF DESULFOFERRODOXIN FROM DESULFOVIBRIO-DESULFURICANS (ATCC-27774)}, journal = {Journal Of Biological Chemistry}, volume = {{269}}, number = {{14}}, year = {1994}, note = {n/a}, pages = {{10504-10510}}, abstract = {

Desulfoferrodoxin, a non-heme iron protein, was purified previously from extracts of Desulfovibrio desulfuricans (ATCC 27774) (Moura, I., Tavares, P., Moura, J. J. G., Ravi, N., Huynh, B. H., Liu, M.-Y., and LeGall, J. (1990) J. Biol. Chem. 265, 21596-21602). The as-isolated protein displays a pink color (pink form) and contains two mononuclear iron sites in different oxidation states: a ferric site (center I) with a distorted tetrahedral sulfur coordination similar to that found in desulforedoxin from Desulfovibrio gigas and a ferrous site (center II) octahedrally coordinated with predominantly nitrogen/ oxygen-containing ligands. A new form of desulfoferrodoxin which displays a gray color (gray form) has now been purified. Optical, electron paramagnetic resonance (EPR), and Mossbauer data of the gray desulfoferrodoxin indicate that both iron centers are in the high-spin ferric states. In addition to the EPR signals originating from center I at g = 7.7, 5.7, 4.1, and 1.8, the gray form of desulfoferrodoxin exhibits a signal atg = 4.3 and a shoulder at g = 9.6, indicating a high-spin ferric state with E/D approximate to 1/3 for the oxidized center II. Redox titrations of the gray form of the protein monitored by optical spectroscopy indicate midpoint potentials of +4 +/- 10 and +240 +/- 10 mV for centers I and II, respectively. Mossbauer spectra of the gray form of the protein are consistent with the EPR finding that both centers are high-spin ferric and can be analyzed in terms of the EPR-determined spin Hamiltonian parameters. The Mossbauer parameters for both the ferric and ferrous forms of center II are indicative of a mononuclear high spin iron site with octahedral coordination and predominantly nitrogen/oxygen-containing ligands. Resonance Raman studies confirm the structural similarity of center I and the distorted tetrahedral FeS4 center in desulforedoxin and provide evidence for one or two cysteinyl-S ligands for center II. On the basis of the resonance Raman results, the 635 nm absorption band that is responsible for the gray color of the oxidized protein is assigned to a cysteinyl-S --\> Fe(III) charge transfer transition localized on center II. The novel properties and possible function of center II are discussed in relation to those of mononuclear iron centers in other enzymes.

}, author = {Tavares, P and Ravi, N. and Moura, J. J. G. and Legall, J. and Huang, Y. H. and Crouse, B. R. and Johnson, M. K. and Huynh, B. H. and Moura, I.} } @article {5384, title = {DIRECT SPECTROSCOPIC EVIDENCE FOR THE PRESENCE OF A 6FE CLUSTER IN AN IRON-SULFUR PROTEIN ISOLATED FROM DESULFOVIBRIO-DESULFURICANS (ATCC-27774)}, journal = {Journal Of Biological Chemistry}, volume = {{267}}, number = {{7}}, year = {1992}, note = {n/a}, pages = {{4489-4496}}, abstract = {

A novel iron-sulfur protein was purified from the extract of Desulfovibrio desulfuricans (ATCC 27774) to homogeneity as judged by polyacrylamide gel electrophoresis. The purified protein is a monomer of 57 kDa molecular mass. It contains comparable amounts of iron and inorganic labile sulfur atoms and exhibits an optical spectrum typical of iron-sulfur proteins with maxima at 400, 305, and 280 nm. Mossbauer data of the as-isolated protein show two spectral components, a paramagnetic and a diamagnetic, of equal intensity. Detailed analysis of the paramagnetic component reveals six distinct antiferromagnetically coupled iron sites, providing direct spectroscopic evidence for the presence of a 6Fe cluster in this newly purified protein. One of the iron sites exhibits parameters (DELTA-E(Q) = 2.67 +/- 0.03 mm/s and delta = 1.09 +/- 0.02 mm/s at 140 K) typical for high spin ferrous ion; the observed large isomer shift indicates an iron environment that is distinct from the tetrahedral sulfur coordination commonly observed for the iron atoms in iron-sulfur clusters and is consistent with a penta- or hexacoordination containing N and/or O ligands. The other five iron sites are most probably high spin ferric. Three of them show parameters characteristic for tetrahedral sulfur coordination. In correlation with the EPR spectrum of the as-purified protein which shows a resonance signal at g = 15.3 and a group of signals between g = 9.8 and 5.4, this 6Fe cluster is assigned to an unusual spin state of 9/2 with zero field splitting parameters D = -1.3 cm-1 and E/D = 0.062. Other EPR signals attributable to minor impurities are also observed at the g = 4.3 and 2.0 regions. The diamagnetic Mossbauer component represents a second iron cluster, which, upon reduction with dithionite, displays an intense S = 1/2 EPR signal with g values at 2.00, 1.83, and 1.31. In addition, an EPR signal of the S = 3/2 type is also observed for the dithionite-reduced protein.

}, author = {Moura, I. and Tavares, P and Moura, J. J. G. and Ravi, N. and Huynh, B. H. and Liu, M. Y. and Legall, J.} } @article {5385, title = {PURIFICATION AND CHARACTERIZATION OF DESULFOFERRODOXIN - A NOVEL PROTEIN FROM DESULFOVIBRIO-DESULFURICANS (ATCC-27774) AND FROM DESULFOVIBRIO-VULGARIS (STRAIN HILDENBOROUGH) THAT CONTAINS A DISTORTED RUBREDOXIN CENTER AND A MONONUCLEAR FERROUS CENTER}, journal = {Journal Of Biological Chemistry}, volume = {{265}}, number = {{35}}, year = {1990}, note = {n/a}, pages = {{21596-21602}}, abstract = {n/a}, author = {Moura, I. and Tavares, P and Moura, J. J. G. and Ravi, N. and Huynh, B. H. and Liu, M. Y. and Legall, J.} }