Adenylyl sulfate (APS) reductase, the key enzyme of the dissimilatory sulfate respiration, catalyzes the reduction of APS (the activated form of sulfate) to sulfite with release of AMP. A spectroscopic study was carried out with the APS reductase purified from the extremely thermophilic sulfate-reducing archaebacterium Archaeoglobus fulgidus DSM 4304. Combined ultraviolet/visible spectroscopy and low temperature electron paramagnetic resonance (EPR) studies were used in order to characterize the active centers and the reactivity towards AMP and sulfite of this enzyme. The A. fulgidus APS reductase is an iron-sulfur flavoprotein containing two distinct [4Fe-4S] clusters (Centers I and II) very similar to the homologous enzyme from Desulfovibrio gigas. Center I, which has a high redox potential, is reduced by AMP and sulfite, and Center II has a very negative redox potential.

A new non-heme iron protein from the periplasmic fraction of Desulfovibrio vulgaris (Hildenbourough NCIB 8303) has been purified to homogeneity, and its amino acid composition, molecular weight, redox potential, iron content, and optical, EPR, and Mossbauer spectroscopic properties have been determined. This new protein is composed of two identical subunits with subunit molecular weight of 21,900 and contains four iron atoms per molecule. The as-purified oxidized protein exhibits an optical spectrum with absorption maxima at 492, 365, and 280 nm, and its EPR spectrum shows resonances at g = 4.3 and 9.4, characteristic of oxidized rubredoxin. The Mossbauer data indicate the presence of approximately equal amounts of two types of iron; we named them the Rd-like and the Hr-like iron due to their similarity to the iron centers of rubredoxins (Rds) and hemerythrins (Hrs), respectively. For the Rd-like iron, the measured fine and hyperfine parameters (D = 1.5 cm-1, E/D = 0.26, delta EQ = -0.55 mm/s, delta = 0.27 mm/s, Axx/gn beta n = -16.5 T, Ayy/gn beta n = -15.6 T, and Azz/gn beta n = -17.0 T) are almost identical with those obtained for the rubredoxin from Clostridium pasteurianum. Redox-titration studies monitored by EPR, however, showed that these Rd-like centers have a midpoint redox potential of +230 +/- 10 mV, approximately 250 mV more positive than those reported for rubredoxins. Another unusual feature of this protein is the presence of the Hr-like iron atoms.(ABSTRACT TRUNCATED AT 250 WORDS)

Three multiheme c-type cytochromes--the tetraheme cytochrome c3 (molecular weight [MW] 13,500), a dodecaheme cytochrome c (MW 40,800), and a "split-Soret" cytochrome c (MW 51,540), which is a dimer with 2 hemes per subunit (MW 26,300)--were isolated from the soluble fraction of Desulfovibrio desulfuricans (ATCC 27774) grown under nitrate- or sulfate-respiring conditions. Two of them, the dodecaheme and the split-Soret cytochromes, showed no similarities to any of the c-type cytochromes isolated from other sulfate-reducing bacteria, while the tetraheme cytochrome c3 appeared to be analogous to the cytochrome c3 found in other sulfate-reducing bacteria. For all three multiheme c-type cytochromes isolated, the homologous proteins from nitrate- and sulfate-grown cells were indistinguishable in amino acid composition, physical properties, and spectroscopic characteristics. It therefore appears that the same c-type cytochrome components are present when D. desulfuricans ATCC 27774 cells are grown under either condition. This is in contrast to the considerable difference found in Pseudomonas perfectomarina (Liu et al., J. Bacteriol. 154:278-286, 1983), a marine denitrifier, when the cells are grown on nitrate or oxygen as the terminal electron acceptor. In addition, two spectroscopy methods capable of revealing minute structural variations in proteins provided identical information about the tetraheme cytochrome c3 from nitrate-grown and sulfate-grown cells.

The recently discovered [2Fe-2S] cluster in mouse liver ferrochelatase has been characterized using UV-vis, EPR, and Mossbauer spectroscopic techniques. Studies are reported here for the recombinant protein purified from an overproducing transformed Escherichia coli strain. A positive correlation is observed between the presence of the [2Fe-2S] cluster and the enzymatic specific activity and demonstrates the necessity of this cofactor. Chemical analysis revealed that the preparations contained up to 1.3 Fe/molecule and indicated a 1:1 stoichiometry between Fe and acid-labile sulfide. The [2Fe-2S] cluster in the as-isolated ferrochelatase exhibits a UV-vis spectrum indicative of a [2Fe-2S](2+) cluster and is EPR-silent. The 8 T Mossbauer spectrum of the Fe-57-enriched as-isolated protein is well simulated by parameters Delta E(Q) = 0.69 +/- 0.03 mm/s and delta = 0.28 +/- 0.02 mm/s and confirms the presence of a diamagnetic ground state. Upon reduction with sodium dithionite, ferrochelatase shows a near-axial EPR spectrum with g-values of 2.00, 1.93, and 1.91, consistent with a S = 1/2 mixed valent Fe3+-Fe2+ cluster. The Orbach temperature dependence of the EPR line widths was used to provide an estimate of the exchange coupling J, which was determined to be on the order of 500-650 cm(-1) (+JS(1) . S-2 model). Redox titrations monitored by UV-vis and EPR spectroscopy revealed midpoint potentials of -390 +/- 10 and -405 +/- 10 mV, respectively. Mossbauer spectra of the sodium dithionite-reduced Fe-57-enriched ferrochelatase collected at 4.2 K in the presence of magnetic fields of 60 mT and 8 T strengths were analyzed in the mixed-valent S = 1/2 ground state. Parameters for the ferric site are Delta E(Q) = 1.2 +/- 0.2 mm/s and delta = 0.28 +/- 0.03 mm/s, with somewhat anisotropic hyperfine splittings; for the ferrous site, Delta E(Q) = 3.3 +/- 0.1 mm/s and delta = 0.67 +/- 0.04 mm/s with anisotropic hyperfine splittings characteristic of high-spin ferrous ion. The similarities and differences with other characterized [2Fe-2S](+) cluster-containing proteins are discussed.

The intermolecular electron transfer kinetics between nitrite reductase (NiR, cytochrome cd1) isolated from Pseudomonas nautica and three cytochromes c isolated from the same strain, as well as the intramolecular electron transfer between NiR heme c and NiR heme d1, were investigated by cyclic voltammetry. All cytochromes (cytochrome c552, cytochrome c553 and cytochrome C553(548)) exhibited well-behaved electrochemistry. The individual diffusion coefficients and mid-point redox potentials were determined. Under the experimental conditions, only cytochrome c552 established a rapid electron transfer with NiR. At acidic pH, the intermolecular electron transfer (cytochrome c(552red)-->NiR heme cox) is a second-order reaction with a rate constant (k2) of 4.1+/-0.1x10(5) M(-1) s(-1) (pH=6.3 and 100 mM NaCl). Under these conditions, the intermolecular reaction represents the rate-limiting step. A minimum estimate of 33 s(-1) could be determined for the first-order rate constant (k1) of the intramolecular electron transfer reaction NiR heme c(red)-->NiR heme d1ox. The pH dependence of k2 values was investigated at pH values ranging from 5.8 to 8.0. When the pH is progressively shifted towards basic values, the rate constant of the intramolecular electron transfer reaction NiR heme c(red)-->NiR heme d1ox decreases gradually to a point where it becomes rate limiting. At pH 8.0 we determined a value of 1.4+/-0.7 s(-1), corresponding to a k2 value of 2.2+/-1.1x10(4) M(-1) s(-1) for the intermolecular step. The physiological relevance of these results is discussed with a particular emphasis on the proposed mechanism of "dead-end product" formation.

The di-haem cytochrome c peroxidase of Paracoccus denitrificans is a calcium binding dimer of 37.5 kDa subunits. It is responsible for reduction of H(2)O(2) to H(2)O with oxidation of cytochrome c(550) and is isolated in a fully oxidised state (inactive) in which one haem (centre I) is in a high-spin/low-spin equilibrium and high potential and the other (centre II) is low-spin and low potential. The enzyme undergoes direct electron transfer (without the need for mediators) with a 4,4'-dithiodipyridine-modified gold electrode and the response of both haem groups can be observed. By combination of the cyclic and pulse voltammetric data with the established spectroscopic information, it was demonstrated that entry of one electron to the high potential haem leads (in a mechanism involving strong haem-haem interactions) to a complex change of spin states and redox potentials of both haems in order to attain a "ready state" for binding, reduction and cleavage of the hydrogen peroxide. In the absence of endogenous calcium, haem communication can be completely disconnected and is recovered only when Ca(2+) is added, an essential step for the formation of the peroxidatic site. The intricate electrochemical behaviour of this enzyme was interpreted as a mechanism involving, both reduction and oxidation of the high potential haem, an interfacial electron transfer coupled to a homogenous chemical reaction (EC mechanism). We discuss two different models for the sequence of events leading to the appearance of the active pentacoordinated peroxidatic haem.

Two proteins were purified and preliminarily characterized from the soluble extract of cells (310 g, wet weight) of the aminolytic and peptidolytic sulfate-reducing bacterium Desulfovibrio (D.) aminophilus DSM 12254. The iron-sulfur flavoenzyme adenylylsulfate (adenosine 5'-phosphosulfate, APS) reductase, a key enzyme in the microbial dissimilatory sulfate reduction, has been purified in three chromatographic steps (DEAE-Biogel A, Source 15, and Superdex 200 columns). It contains two different subunits with molecular masses of 75 and 18 kDa. The fraction after the last purification step had a purity index (A(278nm) / A(388nm)) of 5.34, which was used for further EPR spectroscopic studies. The D. aminophilus APS reductase is very similar to the homologous enzymes isolated from D. gigas and D. desulfuricans ATCC 27774. A tetraheme cytochrome c(3) (His-heme iron-His) has been purified in three chromatographic steps (DEAE- Biogel A, Source 15, and Biogel-HTP columns) and preliminarily characterized. It has a purity index ([A(553nm) - A(570nm)](red) / A(280nm)) of 2.9 and a molecular mass of around 15 kDa, and its spectroscopic characterization (NMR and EPR) has been carried out. This hemoprotein presents similarities with the tetraheme cytochrome c(3) from Desulfomicrobium (Des.) norvegicum (NMR spectra, and N-terminal amino acid sequence).

Rapid identification of proteins is of primary importance for the analytical community. Protein-biomarker discovery for medical diagnostics or pharmacological purposes is becoming one of the hottest research topics. Moreover, rapid identification of proteins can help unambiguous bacterial and virus detection. In addition, the fast identification of bacteria can be used to beat bioterrorism. As a consequence, new analytical methodologies have emerged recently with the aim of making protein analysis as fast and as confident as possible. In this article, we critically review the new trends in sample treatment for protein identification and comment on the prospects for the future in this promising analytical area. (c) 2006 Elsevier Ltd. All rights reserved.

Resonance Raman (RR) spectroscopy is used to examine porphyrin substrate, product, and inhibitor interactions with the active site of murine ferrochelatase (EC 4.99.1.1), the terminal enzyme in the biosynthesis of heme. The enzyme catalyzes in vivo Fe2+ chelation into protoporphyrin IX to give heme. The RR spectra of native ferrochelatase show that the protein, as isolated, contains varying amounts of endogenously bound high- or low-spin ferric heme, always at much less than 1 equiv. RR data on the binding of free-base protoporphyrin IX and its metalated complexes (Fe(III), Fe(II), and Ni(II)) to active wild-type protein were obtained at varying ratios of porphyrin to protein. The binding of ferric heme, a known inhibitor of the enzyme, leads to the formation of a low-spin six-coordinate adduct. Ferrous heme, the enzyme's natural product, binds in the ferrous high-spin five-coordinate state. Ni(II) protoporphyrin, a metalloporphyrin that has a low tendency toward axial ligation, becomes distorted when bound to ferrochelatase. Similarly for free-base protoporphyrin, the natural substrate of ferrochelatase, the RR spectra of porphyrin-protein complexes reveal a saddling distortion of the porphyrin. These results corroborate and extend our previous findings that porphyrin distortion, a crucial step of the catalytic mechanism, occurs even in the absence of bound metal substrate. Moreover, RR data reveal the presence of an amino acid residue in the active site of ferrochelatase which is capable of specific axial ligation to metals.

Resonance Raman (RR) spectroscopy, molecular mechanics (MM) calculations, and normal-coordinate structural decomposition (NSD) have been used to investigate the conformational differences in the hemes in ferricytochromes c3. NSD analyses of heme structures obtained from X-ray crystallography and MM calculations of heme-peptide fragments of the cytochromes c3 indicate that the nonplanarity of the hemes is largely controlled by a fingerprint peptide segment consisting of two heme-linked cysteines, the amino acids between the cysteines, and the proximal histidine ligand. Additional interactions between the heme and the distal histidine ligand and between the heme propionates and the protein also influence the heme conformation, but to a lesser extent than the fingerprint peptide segment. In addition, factors that influence the folding pattern of the fingerprint peptide segment may have an effect on the heme conformation. Large heme structural differences between the baculatum cytochromes c3 and the other proteins are uncovered by the NSD procedure [Jentzen, W., Ma, J.-G., and Shelnutt, J. A. (1998) Biophys. J. 74, 753-763]. These heme differences are mainly associated with the deletion of two residues in the covalently linked segment of hemes 4 for the baculatum proteins. Furthermore, some of these structural differences are reflected in the RR spectra. For example, the frequencies of the structure-sensitive lines (nu4, nu3, and nu2) in the high-frequency region of the RR spectra are lower for the Desulfomicrobium baculatum cytochromes c3 (Norway 4 and 9974) than for the Desulfovibrio (D.) gigas, D. vulgaris, and D. desulfuricans strains, consistent with a more ruffled heme. Spectral decompositions of the nu3 and nu10 lines allow the assignment of the sublines to individual hemes and show that ruffling, not saddling, is the dominant factor influencing the frequencies of the structure-sensitive Raman lines. The distinctive spectra of the baculatum strains investigated are a consequence of hemes 2 and 4 being more ruffled than is typical of the other proteins.

1D and 2D 1H NMR studies are reported on the oxidized and reduced [4Fe-4S] cluster of Desulfovibrio gigas ferredoxin I (Fdl). Several low-field contact shifted resonances (fast relaxing) are assigned to β-CH2 and α-CH coordinated cysteinyl residues. NOESY patterns (supported by 1D NOE experiments) resolves four pairs of geminal β-CH2 protons at low-field. The cluster ligands are assigned non-specifically to Cys8, Cys11, Cys14 and Cys50, based on the X-ray structural analysis available for the oligomeric form, FdII, that contains a single [3Fe-4S] cluster. It was indicated in this case that Cys11 is not bound to the trinuclear cluster but is tilted towards the solvent. The presence of four pairs of geminal β-CH2 protons for FdI unambiguously proves the occupancy of the fourth site of the [3Fe-4S] complex and implies the coordination of the Cys11 at the cluster. Analysis of the oxidized form of FdII, using the same methodology as described for FdI, supports the presence of three cysteinyl ligands in the [3Fe-4S] core. Further, the combined use of the X-ray coordinates enables the specific assignment of the three cysteinyl ligands of the cluster, extending a previous assignment of Cys50. In addition, very broad resonances were detected for the reduced form of FdII in the low-field region around 200 ppm and in the high field region around −80 ppm.

Desulfovibrio gigas ferredoxin II (FdII) is a small protein (alpha 4 subunit structure as isolated; M(r) approximately 6400 per subunit; 6 cysteine residues) containing one Fe3S4 cluster per alpha-subunit. The x-ray structure of FdII has revealed a disulfide bridge formed by Cys-18 and Cys-42 approximately 13 A away from the center of the cluster; moreover, the x-ray structure indicates that Cys-11 forms a disulfide bridge with a methanethiol. In the oxidized state, FdIIoxm the 1H NMR spectra, exhibit four low-field contact-shifted resonances at 29, 24, 18, and 15.5 ppm whereas the reduced state, FdIIR (S = 2), yields two features at +18.5 and -11 ppm. In the course of studying the redox behavior of FdII, we have discovered a stable intermediate, FdIIint, that yields 1H resonances at 24, 21.5, 21, and 14 ppm. This intermediate appears in the potential range where the cluster (E'0 approximately -130 mV) is reduced from the [Fe3S4]1+ to the [Fe3S4]0 state. FdIIint is observed during reductive titrations with dithionite or hydrogen/hydrogenase or after partial oxidation of FdIIR by 2,6-dichlorophenolindophenol or air. Our studies show that a total of three electrons per alpha-subunit are transferred to FdII. Our experiments demonstrate the absence of a methanethiol-Cys-11 linkage in our preparations, and we propose that two of the three electrons are used for the reduction of the disulfide bridge. Mossbauer (and EPR) studies show that the Fe3S4 cluster of FdIIint is at the same oxidation level as FdIIox, but indicate some changes in the exchange couplings among the three ferric sites. Our data suggest that the differences in the NMR and Mossbauer spectra of FdIIox and FdIIint result from conformational changes attending the breaking or formation of the disulfide bridge. The present study suggests that experiments be undertaken to explore an in vivo redox function for the disulfide bridge.

A 7Fe ferredoxin, isolated from the marine denitrifier Pseudomonas nautica strain 617, was characterized. The NH2-terminal sequence analysis, performed until residue number 56, shows a high similarity with the 7Fe ferredoxins isolated from Azotobacter vinelandii, Pseudomonas putida, and Pseudomonas stutzeri. EPR and NMR spectroscopies identify the presence of both [3Fe-4S] and [4Fe-4S] clusters, with cysteinyl coordination. The electrochemical studies on [Fe-S] clusters show that a fast diffusion-dominated electron transfer, promoted by Mg(II), takes place between the ferredoxin and the glassy carbon electrode. Square wave voltammetry studies gave access to the electrosynthesis of a 4Fe center formed within the [3Fe-4S] core. The [3Fe-4S] cluster exhibited two reduction potentials at -175 and -680 +/- 10 mV and the [4Fe-4S] cluster was characterized by an unusually low reduction potential of -715 +/- 10 mV, at pH 7.6

The biological relevance of molybdenum was demonstrated in the early 1950s-1960s, by Bray, Beinert, Lowe, Massey, Palmer, Ehrenberg, Pettersson, Vänngård, Hanson and others, with ground-breaking studies performed, precisely, by electron paramagnetic resonance (EPR) spectroscopy. Those earlier studies, aimed to investigate the mammalian xanthine oxidase and avian sulfite oxidase enzymes, demonstrated the surprising biological reduction of molybdenum to the paramagnetic Mo⁵⁺. Since then, EPR spectroscopy, alongside with other spectroscopic methods and X-ray crystallography, has contributed to our present detailed knowledge about the active site structures, catalytic mechanisms and structure/activity relationships of the molybdenum-containing enzymes.
This Chapter will provide a perspective on the contribution that EPR spectroscopy has made to some selected systems. After a brief overview on molybdoenzymes, the Chapter will be focused on the EPR studies of mammalian xanthine oxidase, with a brief account on the prokaryotic aldehyde oxidoreductase, nicotinate dehydrogenase and carbon monoxide dehydrogenase, vertebrate sulfite oxidase, and prokaryotic formate dehydrogenases and nitrate reductases.

Mammalian xanthine oxidase (XO) and Desulfovibrio gigas aldehyde oxidoreductase (AOR) are members of the XO family of mononuclear molybdoenzymes that catalyse the oxidative hydroxylation of a wide range of aldehydes and heterocyclic compounds. Much less known is the XO ability to catalyse the nitrite reduction to nitric oxide radical (NO). To assess the competence of other XO family enzymes to catalyse the nitrite reduction and to shed some light onto the molecular mechanism of this reaction, we characterised the anaerobic XO- and AOR-catalysed nitrite reduction. The identification of NO as the reaction product was done with a NO-selective electrode and by electron paramagnetic resonance (EPR) spectroscopy. The steady-state kinetic characterisation corroborated the XO-catalysed nitrite reduction and demonstrated, for the first time, that the prokaryotic AOR does catalyse the nitrite reduction to NO, in the presence of any electron donor to the enzyme, substrate (aldehyde) or not (dithionite). Nitrite binding and reduction was shown by EPR spectroscopy to occur on a reduced molybdenum centre. A molecular mechanism of AOR- and XO-catalysed nitrite reduction is discussed, in which the higher oxidation states of molybdenum seem to be involved in oxygen-atom insertion, whereas the lower oxidation states would favour oxygen-atom abstraction. Our results define a new catalytic performance for AOR-the nitrite reduction-and propose a new class of molybdenum-containing nitrite reductases.