Pseudoazurins are small type 1 copper proteins that are involved in the flow of electrons between various electron donors and acceptors in the bacterial periplasm, mostly under denitrifying conditions. The previously determined structure of Paracoccus pantotrophus pseudoazurin in the oxidized form was improved to a nominal resolution of 1.4 angstrom, with R and R(free) values of 0.188 and 0.206, respectively. This high-resolution structure makes it possible to analyze the interactions between the monomers and the solvent structure in detail. Analysis of the high-resolution structure revealed the structural regions that are responsible for monomer-monomer recognition during dimer formation and for protein-protein interaction and that are important for partner recognition. The pseudoazurin structure was compared with other structures of various type 1 copper proteins and these were grouped into families according to similarities in their secondary structure; this may be useful in the annotation of copper proteins in newly sequenced genomes and in the identification of novel copper proteins.

Crystallographic studies of the hydrogenases (Hases) from Desulfovibrio gigas (Dg) and Desulfovibrio vulgaris Miyazaki (DvM) have revealed heterodinuclear nickel-iron active centers in both enzymes. The structures, which represent the as-isolated (unready) Ni-A (S = (1)/(2)) enzyme state, disclose a nonprotein ligand (labeled as X) bridging the two metals. The bridging atom was suggested to be an oxygenic (O(2)(-) or OH(-)) species in Dg Hase and an inorganic sulfide in DvM Hase. To determine the nature and chemical characteristics of the Ni-X-Fe bridging ligand in Dg Hase, we have performed 35 GHz CW (17)O ENDOR measurements on the Ni-A form of the enzyme, exchanged into H(2)(17)O, on the active Ni-C (S = (1)/(2)) form prepared by H(2)-reduction of Ni-A in H(2)(17)O, and also on Ni-A formed by reoxidation of Ni-C in H(2)(17)O. In the native state of the protein (Ni-A), the bridging ligand does not exchange with the H(2)(17)O solvent. However, after a reduction/reoxidation cycle (Ni-A --> Ni-C --> Ni-A), an (17)O label is introduced at the active site, as seen by ENDOR. Detailed analysis of a 2-D field-frequency plot of ENDOR spectra taken across the EPR envelope of Ni-A((17)O) shows that the incorporated (17)O has a roughly axial hyperfine tensor, A((17)O) approximately [5, 7, 20] MHz, discloses its orientation relative to the g tensor, and also yields an estimate of the quadrupole tensor. The substantial isotropic component (a(iso)((17)O) approximately 11 MHz) of the hyperfine interaction indicates that a solvent-derived (17)O is indeed a ligand to Ni and thus that the bridging ligand X in the Ni-A state of Dg Hase is indeed an oxygenic (O(2)(-) or OH(-)) species; comparison with earlier EPR results by others indicates that the same holds for Ni-B. The small (57)Fe hyperfine coupling seen previously for Ni-A (A((57)Fe) approximately 0.9 MHz) is now shown to persist in Ni-C, A((57)Fe) approximately 0.8 MHz. However, the (17)O signal is lost upon reductive activation to the Ni-C state; reoxidation to Ni-A leads to the reappearance of the signal. Consideration of the electronic structure of the EPR-active states of the dinuclear center leads us to suggest that the oxygenic bridge in Ni-A(B) is lost in Ni-C and is re-formed from solvent upon reoxidation to Ni-A. This implies that the reductive activation to Ni-C opens Ni/Fe coordination sites which may play a central role in the enzyme's activity.

In order to utilize sulfate as the terminal electron acceptor, sulfate-reducing bacteria are equipped with a complex enzymatic system in which adenylylsulfate (AdoPSO4) reductase plays one of the major roles, reducing AdoPSO4 (the activated form of sulfate) to sulfite, with release of AMP. The enzyme has been purified to homogeneity from the anaerobic sulfate reducer Desulfovibrio gigas. The protein is composed of two non-identical subunits (70 kDa and 23 kDa) and is isolated in a multimeric form (approximately 400 kDa). It is an iron-sulfur, flavin-containing protein, with one FAD moiety, eight iron atoms and a minimum molecular mass of 93 kDa. Low-temperature EPR studies were performed to characterize its redox centers. In the native state, the enzyme showed an almost isotropic signal centered at g = 2.02 and only detectable below 20 K. This signal represented a minor species (0.10-0.25 spins/mol) and showed line broadening in the enzyme isolated from 57Fe-grown cells. Addition of sulfite had a minor effect on the EPR spectrum, but caused a major decrease in the visible region of the optical spectrum (around 392 nm). Further addition of AMP induced only a minor change in the visible spectrum whereas major changes were seen in the EPR spectrum; the appearance of a rhombic signal at g values 2.096, 1.940 and 1.890 (reduced Fe-S center I) observable below 30 K and a concomitant decrease in intensity of the g = 2.02 signal were detected. Effects of chemical reductants (ascorbate, H2/hydrogenase-reduced methyl viologen and dithionite) were also studied. A short time reduction with dithionite (15 s) or reduction with methyl viologen gave rise to the full reduction of center I (with slightly modified g values at 2.079, 1.939 and 1.897), and the complete disappearance of the g = 2.02 signal. Further reduction with dithionite produces a very complex EPR spectrum of a spin-spin-coupled nature (observable below 20 K), indicating the presence of at least two iron-sulfur centers, (centers I and II). Mossbauer studies on 57Fe-enriched D. gigas AdoPSO4 reductase demonstrated unambiguously the presence of two 4Fe clusters. Center II has a redox potential less than or equal to 400 mV and exhibits spectroscopic properties that are characteristic of a ferredoxin-type [4Fe-4S] cluster. Center I exhibits spectra with atypical Mossbauer parameters in its reduced state and has a midpoint potential around 0 mV, which is distinct from that of a ferredoxin-type [4Fe-4S] cluster, suggesting a different structure and/or a distinct cluster-ligand environment.

The binding of Ca2+ to the dihaem cytochrome-c peroxidase from Paracoccus denitrificans was analysed by following perturbations in the visible and 1H-NMR spectra of both haem groups. The enzyme contains at least two types of Ca(2+)-binding site. Site I is occupied in the isolated enzyme, binds Ca2+ with a redox-state-independent Kd of 1.2 microM and accommodates neither Mg2+ nor Mn2+. Site II is unoccupied in dilute solutions of the isolated oxidised enzyme and binds Ca2+ cooperatively with a Kd of 0.52 mM. In the mixed valence form, the binding affinity increases to resemble that of site I. The cooperativity was shown by -Ca2+ binding to site II, the titration of haem methyl 1H-NMR resonances, and a half-of-sites effect observed for modification of an essential histidine with diethylpyrocarbonate. These are all consistent with site II being situated at the interface between two monomers of a dimeric enzyme. Thus the equilibrium of binding to site II is a reflection of the equilibrium for dimerisation and conditions which shift that equilibrium towards the dimer, such as increased ionic strength or high protein concentration, also increase Ca2+ affinity. Binding of Ca2+ to site II is required for formation of the active high spin state at the peroxidatic haem.

Analysis of sequenced bacterial genomes revealed that the genomes encode more than 30% hypothetical and conserved hypothetical proteins of unknown function. Among proteins of unknown function that are conserved in anaerobes, some might be determinants of the anaerobic way of life. This study focuses on two divergent clusters specifically found in anaerobic microorganisms and mainly composed of genes encoding conserved hypothetical proteins. We show that the two gene clusters DVU2103-DVU2104-DVU2105 (orp2) and DVU2107-DVU2108-DVU2109 (orp1) form two divergent operons transcribed by the sigma(54)-RNA polymerase. We further demonstrate that the sigma(54)-dependent transcriptional regulator DVU2106, located between orp1 and orp2, collaborates with sigma(54)-RNA polymerase to orchestrate the simultaneous expression of the divergent orp operons. DVU2106, whose structural gene is transcribed by the sigma(70)-RNA polymerase, negatively retrocontrols its own expression. By using an endogenous pulldown strategy, we identify a physiological complex composed of DVU2103, DVU2104, DVU2105, DVU2108, and DVU2109. Interestingly, inactivation of DVU2106, which is required for orp operon transcription, induces morphological defects that are likely linked to the absence of the ORP complex. A putative role of the ORP proteins in positioning the septum during cell division is discussed.

The activation mechanism of Pseudomonas stutzeri cytochrome c peroxidase (CCP) was probed through the mediated electrochemical catalysis by its physiological electron donor, P. stutzeri cytochrome c-551. A comparative study was carried out, by performing assays with the enzyme in the resting oxidized state as well as in the mixed-valence activated form, using cyclic voltammetry and a pyrolytic graphite membrane electrode. In the presence of both the enzyme and hydrogen peroxide, the peak-like signal of cytochrome c-551 is converted into a sigmoidal wave form characteristic of an E(r)C'(i) catalytic mechanism. An intermolecular electron transfer rate constant of (4 +/- 1) x 10(5) M(-1) s(-1) was estimated for both forms of the enzyme, as well as a similar Michaelis-Menten constant. These results show that neither the intermolecular electron transfer nor the catalytic activity is kinetically controlled by the activation mechanism of CCP in the case of the P. stutzeri enzyme. Direct enzyme catalysis using protein film voltammetry was unsuccessful for the analysis of the activation mechanism, since P. stutzeri CCP undergoes an undesirable interaction with the pyrolytic graphite surface. This interaction, previously reported for the Paracoccus pantotrophus CCP, induces the formation of a non-native conformation state of the electron-transferring haem, which has a redox potential 200 mV lower than that of the native state and maintains peroxidatic activity.

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 [S(2)MoS(2)CuS(2)MoS(2)] [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 MoS(4)(2-) 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.

In this work it is demonstrated that the characterization of c-type haem containing proteins by electrochemical techniques needs to be cautiously performed when using pyrolytic graphite electrodes. An altered form of the cytochromes, which has a redox potential 300 mV lower than that of the native state and displays peroxidatic activity, can be induced by interaction with the pyrolytic graphite electrode. Proper control experiments need to be performed, as altered conformations of the enzymes containing c-type haems can show activity towards the enzyme substrate. The work was focused on the study of the activation mechanism and catalytic activity of cytochrome c peroxidase from Paracoccus pantotrophus. The results could only be interpreted with the assignment of the observed non-turnover and catalytic signals to a non-native conformation state of the electron-transferring haem. The same phenomenon was detected for Met-His monohaem cytochromes (mitochondrial cytochrome c and Desulfovibrio vulgaris cytochrome c-553), as well as for the bis-His multihaem cytochrome c(3) from Desulfovibrio gigas, showing that this effect is independent of the axial coordination of the c-type haem protein. Thus, the interpretation of electrochemical signals of c-type (multi)haem proteins at pyrolytic graphite electrodes must be carefully performed, to avoid misassignment of the signals and incorrect interpretation of catalytic intermediates.

A comparative study of direct and mediated electrochemistry of metalloproteins in bulk and membrane-entrapped solutions is presented. This work reports the first electrochemical study of the electron transfer between a bacterial cytochrome c peroxidase and horse heart cytochrome c. The mediated catalysis of the peroxidase was analysed both using the membrane electrode configuration and with all proteins in solution. An apparent Michaelis constant of 66 +/- 4 and 42 +/- 5 microM was determined at pH 7.0 and 0 M NaCl for membrane and bulk solutions, respectively. The data revealed that maximum activity occurs at 50 mM NaCl, pH 7.0, with intermolecular rate constants of (4.4 +/- 0.5) x 10(6) and (1.0 +/- 0.5) x 10(6) M(-1) s(-1) for membrane-entrapped and bulk solutions, respectively. The influence of parameters such as pH or ionic strength on the mediated catalytic activity was analysed using this approach, drawing attention to the fact that careful analysis of the results is needed to ensure that no artefacts are introduced by the use of the membrane configuration and/or promoters, and therefore the dependence truly reflects the influence of these parameters on the (mediated) catalysis. From the pH dependence, a pK of 7.5 was estimated for the mediated enzymatic catalysis.

A new computationally efficient and automated "soft docking" algorithm is described to assist the prediction of the mode of binding between two proteins, using the three-dimensional structures of the unbound molecules. The method is implemented in a software package called BiGGER (Bimolecular Complex Generation with Global Evaluation and Ranking) and works in two sequential steps: first, the complete 6-dimensional binding spaces of both molecules is systematically searched. A population of candidate protein-protein docked geometries is thus generated and selected on the basis of the geometric complementarity and amino acid pairwise affinities between the two molecular surfaces. Most of the conformational changes observed during protein association are treated in an implicit way and test results are equally satisfactory, regardless of starting from the bound or the unbound forms of known structures of the interacting proteins. In contrast to other methods, the entire molecular surfaces are searched during the simulation, using absolutely no additional information regarding the binding sites. In a second step, an interaction scoring function is used to rank the putative docked structures. The function incorporates interaction terms that are thought to be relevant to the stabilization of protein complexes. These include: geometric complementarity of the surfaces, explicit electrostatic interactions, desolvation energy, and pairwise propensities of the amino acid side chains to contact across the molecular interface. The relative functional contribution of each of these interaction terms to the global scoring function has been empirically adjusted through a neural network optimizer using a learning set of 25 protein-protein complexes of known crystallographic structures. In 22 out of 25 protein-protein complexes tested, near-native docked geometries were found with C(alpha) RMS deviations < or =4.0 A from the experimental structures, of which 14 were found within the 20 top ranking solutions. The program works on widely available personal computers and takes 2 to 8 hours of CPU time to run any of the docking tests herein presented. Finally, the value and limitations of the method for the study of macromolecular interactions, not yet revealed by experimental techniques, are discussed.

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 sulfido-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.

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.

The structural changes in the heme macrocycle and substituents caused by binding of Ca2+ to the diheme cytochrome c peroxidase from Paracoccus pantotrophus were clarified by resonance Raman spectroscopy of the inactive fully oxidized form of the enzyme. The changes in the macrocycle vibrational modes are consistent with a Ca2+-dependent increase in the out-of-plane distortion of the low-potential heme, the proposed peroxidatic heme. Most of the increase in out-of-plane distortion occurs when the high-affinity site I is occupied, but a small further increase in distortion occurs when site II is also occupied by Ca2+ or Mg2+. This increase in the heme distortion explains the red shift in the Soret absorption band that occurs upon Ca2+ binding. Changes also occur in the low-frequency substituent modes of the heme, indicating that a structural change in the covalently attached fingerprint pentapeptide of the LP heme occurs upon Ca2+ binding to site I. These structural changes may lead to loss of the sixth ligand at the peroxidatic heme in the semireduced form of the enzyme and activation.

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). 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.

Redox intermediates of D. desulfuricans ATCC 27774 [NiFe] hydrogenase were generated under dihydrogen. Detailed redox titrations, coupled to EPR measurements, give access to the mid-point redox potentials of the iron-sulfur centers and of the Nickel-B signal that represents the ready form of the enzyme. The interaction between the dihydrogen molecule and the nickel centre was probed by the observation of an isotopic effect on the EPR signals detected in turnover conditions, by comparison of the H2O/H2 and D2O/D2-reacted samples.

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.

We have cloned the gene encoding Desulfovibrio gigas ferredoxin using a photodigoxigenin-labelled probe synthesized with the polymerase chain reaction. The DNA sequence of the gene predicts a polypeptide of 58 residues after removal of the initial formyl methionine (polypeptide M(r) = 6,276). The ferredoxin gene was expressed in aerobically grown E. coli behind the lac promoter of pUC18 resulting in a high level of ferredoxin expression which comprises about 10% of the total cell protein. EPR analysis of recombinant ferredoxin revealed the presence of a [3Fe-4S] cluster which is characteristic of native D. gigas ferredoxin II.

Cytochrome c peroxidase from Paracoccus denitrificans LMD 52.44 was recently identified. The enzyme contains two c-type haems: one is reducible physiologically by cytochrome c550 from the same organism or non-physiologically by ascorbate (high-potential haem) and the other by dithionite (low-potential haem). The enzymatically active form of the peroxidase is the half-reduced enzyme state, in which the high-potential haem is in the iron(II) state and the low-potential haem is in the iron(III) state. It was found that the two haems interact and that the enzyme binds calcium ions near the haem sites which are necessary to promote its activation. In the oxidized form, the high-potential haem is in a high-spin and the low-potential haem is in a low-spin state. The half-reduction of the enzyme with ascorbate-diaminodurol changes the high-potential haem (high-spin) into a low-spin state and the low-potential haem converts from a low- into a high-spin state. This high-spin conversion of the low-potential haem is induced by the presence of calcium ions. These processes of reduction and spin state change can be easily resolved in time by removing the calcium from the enzyme using EDTA, facilitating the observation of the intermediate form by NMR.