Probing the dynamic properties of two sites simultaneously in a protein-protein interaction process: a SDSL-EPR study.

During molecular processes, protein flexibility is a fundamental property allowing protein-protein interaction. Following structural changes during these interactions is then of crucial interest. Site-Directed Spin Labeling (SDSL) combined to EPR spectroscopy is a powerful technique to follow structural modifications within proteins and during protein-protein interactions. Usual nitroxide labels target cysteine residues and afford a 3-line spectrum, whose shape is informative of the structural environment of the label. However, it is not possible to probe two regions of a protein or two partner proteins at the same time because of the overlapping of EPR signatures. Previously, we reported the design and the characterization of a spin label based on a ß-phosphorylated (PP) nitroxide yielding a 6-line spectrum. Here, we report the use of two labels with different EPR signatures, namely maleimido-proxyl (P) and PP, to follow structural changes during a protein-protein interaction process in one single experiment. As a model system, we chose a disordered protein that undergoes an induced α-helical folding upon binding to its partner. We show that the EPR spectrum of a mixture of labeled interacting proteins can be analyzed in terms of structural changes during the interaction. This study represents an important step forward in the extension of the panoply of SDSL-EPR approaches.

Hydrogenases in sulfate-reducing bacteria function as chromium reductase.

The ability of sulfate-reducing bacteria (SRB) to reduce chromate VI has been studied for possible application to the decontamination of polluted environments. Metal reduction can be achieved both chemically, by H(2)S produced by the bacteria, and enzymatically, by polyhemic cytochromes c(3). We demonstrate that, in addition to low potential polyheme c-type cytochromes, the ability to reduce chromate is widespread among [Fe], [NiFe], and [NiFeSe] hydrogenases isolated from SRB of the genera Desulfovibrio and Desulfomicrobium. Among them, the [Fe] hydrogenase from Desulfovibrio vulgaris strain Hildenborough reduces Cr(VI) with the highest rate. Both [Fe] and [NiFeSe] enzymes exhibit the same K(m) towards Cr(VI), suggesting that Cr(VI) reduction rates are directly correlated with hydrogen consumption rates. Electron paramagnetic resonance spectroscopy enabled us to probe the oxidation by Cr(VI) of the various metal centers in both [NiFe] and [Fe] hydrogenases. These experiments showed that Cr(VI) is reduced to paramagnetic Cr(III), and revealed inhibition of the enzyme at high Cr(VI) concentrations. The significant decrease of both hydrogenase and Cr(VI)-reductase activities in a mutant lacking [Fe] hydrogenase demonstrated the involvement of this enzyme in Cr(VI) reduction in vivo. Experiments with [3Fe-4S] ferredoxin from Desulfovibrio gigas demonstrated that the low redox [Fe-S] (non-heme iron) clusters are involved in the mechanism of metal reduction by hydrogenases.

Crystal structure of the free radical intermediate of pyruvate:ferredoxin oxidoreductase.

In anaerobic organisms, the decarboxylation of pyruvate, a crucial component of intermediary metabolism, is catalyzed by the metalloenzyme pyruvate: ferredoxin oxidoreductase (PFOR) resulting in the generation of low potential electrons and the subsequent acetylation of coenzyme A (CoA). PFOR is the only enzyme for which a stable acetyl thiamine diphosphate (ThDP)-based free radical reaction intermediate has been identified. The 1.87 A-resolution structure of the radical form of PFOR from Desulfovibrio africanus shows that, despite currently accepted ideas, the thiazole ring of the ThDP cofactor is markedly bent, indicating a drastic reduction of its aromaticity. In addition, the bond connecting the acetyl group to ThDP is unusually long, probably of the one-electron type already described for several cation radicals but not yet found in a biological system. Taken together, our data, along with evidence from the literature, suggest that acetyl-CoA synthesis by PFOR proceeds via a condensation mechanism involving acetyl (PFOR-based) and thiyl (CoA-based) radicals.

The coordination and function of the redox centres of the membrane-bound nitrate reductases.

Under anaerobic conditions and in the presence of nitrate, the facultative anaerobe Escherichia coli synthesises an electron-transport chain comprising a primary dehydrogenase and the terminal membrane-bound nitrate reductase A (NarGHI). This review focuses on recent advances obtained on the structure and function of the three protein subunits of membrane-bound nitrate reductases. We discuss a global architecture for the Mo-bisMGD-containing subunit (NarG) and a coordination model for the four [Fe-S] centres of the electron-transfer subunit (NarH) and for the two b-type haems of the anchor subunit NarI.

Is there a rate-limiting step in the catalytic cycle of Ni-Fe hydrogenases?

The question of the existence of a rate-limiting step in the catalytic cycle of Ni-Fe hydrogenases was taken up by using the sets of data available in the case of two specific enzymes: the hydrogenase from Thiocapsa roseopercisina, in which isotope effects have been systematically investigated over a wide pH range, and the enzyme from Desulfovibrio fructosovorans, for which the activities and the redox properties have been studied in two different forms, the wild type and the P238C mutant. When these data are analyzed in the light of appropriate kinetic models, it is concluded that electron transfer and proton transfer are rate limiting in the H2 uptake and H2 evolution reactions, respectively. This proposal is consistent with the data available from other Ni-Fe enzymes.

Analysis of the electron paramagnetic resonance properties of the [2Fe-2S]1+ centers in molybdenum enzymes of the xanthine oxidase family: assignment of signals I and II.

Molybdoenzymes of the xanthine oxidase family contain two [2Fe-2S](1+,2+) clusters that are bound to the protein by very different cysteine motifs. In the X-ray crystal structure of Desulfovibrio gigas aldehyde oxidoreductase, the cluster ligated by a ferredoxin-type motif is close to the protein surface, whereas that ligated by an unusual cysteine motif is in contact with the molybdopterin [Romao, M. J., Archer, M., Moura, I., Moura, J. J. G., LeGall, J., Engh, R., Schneider, M., Hof, P., and Huber, R. (1995) Science 270, 1170-1176]. These two clusters display distinct electron paramagnetic resonance (EPR) signals: the less anisotropic one, called signal I, is generally similar to the g(av) approximately 1.96-type signals given by ferredoxins, whereas signal II often exhibits anomalous properties such as very large g values, broad lines, and very fast relaxation properties. A detailed comparison of the temperature dependence of the spin-lattice relaxation time and of the intensity of these signals in D. gigas aldehyde oxidoreductase and in milk xanthine oxidase strongly suggests that the peculiar EPR properties of signal II arise from the presence of low-lying excited levels reflecting significant double exchange interactions. The issue raised by the assignment of signals I and II to the two [2Fe-2S](1+) clusters was solved by using the EPR signal of the Mo(V) center as a probe. The temperature dependence of this signal could be quantitatively reproduced by assuming that the Mo(V) center is coupled to the cluster giving signal I in xanthine oxidase as well as in D. gigas aldehyde oxidoreductase. This demonstrates unambiguously that, in both enzymes, signal I arises from the center which is closest to the molybdenum cofactor.

Characterization of a nif-regulated flavoprotein (FprA) from Rhodobacter capsulatus. Redox properties and molecular interaction with a [2Fe-2S] ferredoxin.

A flavoprotein from Rhodobacter capsulatus was purified as a recombinant (His)6-tag fusion from an Escherichia coli clone over-expressing the fprA structural gene. The FprA protein is a homodimer containing one molecule of FMN per 48-kDa monomer. Reduction of the flavoprotein by dithionite showed biphasic kinetics, starting with a fast step of semiquinone (SQ) formation, and followed by a slow reduction of the SQ. This SQ was in the anionic form as shown by EPR and optical spectroscopies. Spectrophotometric titration gave a midpoint redox potential for the oxidized/SQ couple of Em1 = +20 mV (pH 8.0), whereas the SQ/hydroquinone couple could not be titrated due to the thermodynamic instability of SQ associated with its slow reduction process. The inability to detect the intermediate form, SQ, upon oxidative titration confirmed this instability and led to an estimate of Em2 - Em1 of > 80 mV. The reduction of SQ by dithionite was significantly accelerated when the [2Fe-2S] ferredoxin FdIV was used as redox mediator. The midpoint redox potential of this ferredoxin was determined to be -275 +/- 2 mV at pH 7.5, consistent with FdIV serving as electron donor to FprA in vivo. FdIV and FprA were found to cross-react when incubated together with the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, giving a covalent complex with an Mr of approximately 60 000. Formation of this complex was unaffected by the redox states of the two proteins. Other [2Fe-2S] ferredoxins, including FdV and FdVI from R. capsulatus, were ineffective as electron carriers to FprA, and cross-reacted poorly with the flavoprotein. The possible function of FprA with regard to nitrogen fixation was investigated using an fprA-deleted mutant. Although nitrogenase activity was significantly reduced in the mutant compared with the wild-type strain, nitrogen fixation was apparently unaffected by the fprA deletion even under iron limitation or microaerobic conditions.

EPR spectroscopy: a powerful technique for the structural and functional investigation of metalloproteins.

Numerous metal centers in proteins can be prepared in a redox state in which their ground state is paramagnetic. Complementary data provided by EPR, Mössbauer, electron nuclear double resonance, magnetic circular dichroism, and NMR spectroscopies have therefore played a major role in the elucidation of the structure and function of these centers. Among those techniques the most commonly used is certainly EPR spectroscopy. In this article various aspects of the current applications of EPR to the structural and functional study of metalloproteins are presented. They are illustrated by recent studies carried out in our laboratory in the field of metalloenzymes and electron transfer systems. The power of numerical simulation techniques is emphasized throughout this work.

Redox components of cytochrome bc-type enzymes in acidophilic prokaryotes. II. The Rieske protein of phylogenetically distant acidophilic organisms.

The Rieske proteins of two phylogenetically distant acidophilic organisms, i.e. the proteobacterium Thiobacillus ferrooxidans and the crenarchaeon Sulfolobus acidocaldarius, were studied by EPR. Redox titrations at a range of pH values showed that the Rieske centers of both organisms are characterized by redox midpoint potential-versus-pH curves featuring a common pK value of 6.2. This pK value is significantly more acidic (by almost 2 pH units) than that of Rieske proteins in neutrophilic species. The orientations of the Rieske center's g tensors with respect to the plane of the membrane were studied between pH 4 and 8 using partially ordered samples. At pH 4, the Sulfolobus Rieske cluster was found in the "typical" orientation of chemically reduced Rieske centers, whereas this orientation changed significantly on going toward high pH values. The Thiobacillus protein, by contrast, appeared to be in the "standard" orientation at both low and high pH values. The results are discussed with respect to the molecular parameters conveying acid resistance and in light of the recently demonstrated long-range conformational movement of the Rieske protein during enzyme turnover in cytochrome bc1 complexes.

Unusual NMR, EPR, and Mössbauer properties of Chromatium vinosum 2[4Fe-4S] ferredoxin.

The ferredoxin from Chromatium vinosum (CvFd) exhibits sequence and structure peculiarities. Its two Fe4S4(SCys)4 clusters have unusually low potential transitions that have been unambiguously assigned here through NMR, EPR, and Mössbauer spectroscopy in combination with site-directed mutagenesis. The [4Fe-4S]2+/1+ cluster (cluster II) whose coordination sphere includes a two-turn loop between cysteines 40 and 49 was reduced by dithionite with an E degrees ' of -460 mV. Its S = 1/2 EPR signal was fast relaxing and severely broadened by g-strain, and its Mössbauer spectra were broad and unresolved. These spectroscopic features were sensitive to small perturbations of the coordination environment, and they were associated with the particular structural elements of CvFd, including the two-turn loop between two ligands and the C-terminal alpha-helix. Bulk reduction of cluster I (E degrees ' = -660 mV) was not possible for spectroscopic studies, but the full reduction of the protein was achieved by replacing valine 13 with glycine due to an approximately 60 mV positive shift of the potential. At low temperatures, the EPR spectrum of the fully reduced protein was typical of two interacting S = 1/2 [4Fe-4S]1+ centers, but because the electronic relaxation of cluster I is much slower than that of cluster II, the resolved signal of cluster I was observed at temperatures above 20 K. Contact-shifted NMR resonances of beta-CH2 protons were detected in all combinations of redox states. These results establish that electron transfer reactions involving CvFd are quantitatively different from similar reactions in isopotential 2[4Fe-4S] ferredoxins. However, the reduced clusters of CvFd have electronic distributions that are similar to those of clusters coordinated by the CysIxxCysIIxxCysIII.CysIVP sequence motif found in other ferredoxins with different biochemical properties. In all these cases, the electron added to the oxidized clusters is mainly accommodated in the pair of iron ions coordinated by CysII and CysIV.

[3Fe-4S] to [4Fe-4S] cluster conversion in Desulfovibrio fructosovorans [NiFe] hydrogenase by site-directed mutagenesis.

The role of the high potential [3Fe-4S]1+,0 cluster of [NiFe] hydrogenase from Desulfovibrio species located halfway between the proximal and distal low potential [4Fe-4S]2+,1+ clusters has been investigated by using site-directed mutagenesis. Proline 238 of Desulfovibrio fructosovorans [NiFe] hydrogenase, which occupies the position of a potential ligand of the lacking fourth Fe-site of the [3Fe-4S] cluster, was replaced by a cysteine residue. The properties of the mutant enzyme were investigated in terms of enzymatic activity, EPR, and redox properties of the iron-sulfur centers and crystallographic structure. We have shown on the basis of both spectroscopic and x-ray crystallographic studies that the [3Fe-4S] cluster of D. fructosovorans hydrogenase was converted into a [4Fe-4S] center in the P238 mutant. The [3Fe-4S] to [4Fe-4S] cluster conversion resulted in a lowering of approximately 300 mV of the midpoint potential of the modified cluster, whereas no significant alteration of the spectroscopic and redox properties of the two native [4Fe-4S] clusters and the NiFe center occurred. The significant decrease of the midpoint potential of the intermediate Fe-S cluster had only a slight effect on the catalytic activity of the P238C mutant as compared with the wild-type enzyme. The implications of the results for the role of the high-potential [3Fe-4S] cluster in the intramolecular electron transfer pathway are discussed.

Heterologous expression of the Desulfovibrio gigas [NiFe] hydrogenase in Desulfovibrio fructosovorans MR400.

The ability of Desulfovibrio fructosovorans MR400 DeltahynABC to express the heterologous cloned [NiFe] hydrogenase of Desulfovibrio gigas was investigated. The [NiFe] hydrogenase operon from D. gigas, hynABCD, was cloned, sequenced, and introduced into D. fructosovorans MR400. A portion of the recombinant heterologous [NiFe] hydrogenase was totally matured, exhibiting catalytic and spectroscopic properties identical to those of the native D. gigas protein. A chimeric operon containing hynAB from D. gigas and hynC from D. fructosovorans placed under the control of the D. fructosovorans hynAp promoter was constructed and expressed in D. fructosovorans MR400. Under these conditions, the same level of activity was obtained as with the D. gigas hydrogenase operon.

Temperature-jump and potentiometric studies on recombinant wild type and Y143F and Y254F mutants of Saccharomyces cerevisiae flavocytochrome b2: role of the driving force in intramolecular electron transfer kinetics.

The kinetics of intramolecular electron transfer between flavin and heme in Saccharomyces cerevisiae flavocytochrome b2 were investigated by performing potentiometric titrations and temperature-jump experiments on the recombinant wild type and Y143F and Y254F mutants. The midpoint potential of heme was determined by monitoring redox titrations spectrophotometrically, and that of semiquinone flavin/reduced flavin (Fsq/Fred) and oxidized flavin (Fox)/Fsq couples by electron paramagnetic resonance experiments at room temperature. The effects of pyruvate on the kinetic and thermodynamic parameters were also investigated. At room temperature, pH 7.0 and I = 0.1 M, the redox potential of the Fsq/Fred, Fox/Fsq, and oxidized heme/reduced heme (Hox/Hred) couples were -135, -45, and -3 mV, respectively, in the wild-type form. Although neither the mutations nor excess pyruvate did appreciably modify the potential of the heme or that of the Fsq/Fred couple, they led to variable positive shifts in the potential of the Fox/Fsq couple, thus modulating the driving force that characterizes the reduction of heme by the semiquinone in the -42 to +88 mV range. The relaxation rates measured at 16 degreesC in temperature-jump experiments were independent of the protein concentrations, with absorbance changes corresponding to the reduction of the heme. Two relaxation processes were clearly resolved in wild-type flavocytochrome b2 (1/tau1 = 1500 s-1, 1/tau2 = 200 +/- 50 s-1) and were assigned to the reactions whereby the heme is reduced by Fred and Fsq, respectively. The rate of the latter reaction was determined in the whole series of proteins. Its variation as a function of the driving force is well described by the expression obtained from electron-transfer theories, which provides evidence that the intramolecular electron transfer is not controlled by the dynamics of the protein.

NarJ is a specific chaperone required for molybdenum cofactor assembly in nitrate reductase A of Escherichia coli.

The formation of active membrane-bound nitrate reductase A in Escherichia coli requires the presence of three subunits, NarG, NarH and NarI, as well as a fourth protein, NarJ, that is not part of the active nitrate reductase. In narJ strains, both NarG and NarH subunits are associated in an unstable and inactive NarGH complex. A significant activation of this complex was observed in vitro after adding purified NarJ-6His polypeptide to the cell supernatant of a narJ strain. Once the apo-enzyme NarGHI of a narJ mutant has become anchored to the membrane via the NarI subunit, it cannot be reactivated by NarJ in vitro. NarJ protein specifically recognizes the catalytic NarG subunit. Fluorescence, electron paramagnetic resonance (EPR) spectroscopy and molybdenum quantification based on inductively coupled plasma emission spectroscopy (ICPES) clearly indicate that, in the absence of NarJ, no molybdenum cofactor is present in the NarGH complex. We propose that NarJ is a specific chaperone that binds to NarG and may thus keep it in an appropriate competent-open conformation for the molybdenum cofactor insertion to occur, resulting in a catalytically active enzyme. Upon insertion of the molybdenum cofactor into the apo-nitrate reductase, NarJ is then dissociated from the activated enzyme.

Molybdenum cofactor properties and [Fe-S] cluster coordination in Escherichia coli nitrate reductase A: investigation by site-directed mutagenesis of the conserved his-50 residue in the NarG subunit.

Most of the molybdoenzymes contain, in the amino-terminal region of their catalytic subunits, a conserved Cys group that in some cases binds an [Fe-S] cluster. In dissimilatory nitrate reductases, the first Cys residue of this motif is replaced by a conserved His residue. Site-directed mutagenesis of this residue (His-50) was performed on the NarG subunit from Escherichia coli nitrate reductase A. The results obtained by EPR spectroscopy enable us to exclude the implication of this residue in [Fe-S] binding. Additionally, we showed that the His-50 residue does not coordinate the molybdenum atom, but its substitution by Cys or Ser introduces a perturbation of the hydrogen bonding network around the molybdenum cofactor. From potentiometric studies, it is proposed that the high-pH and the low-pH forms of the Mo(V) are both involved during the redox turnover of the enzyme. Perturbation of the Mo(V) pKV value might be responsible for the low activity reported in the His-50-Cys mutant enzyme. A catalytic model is proposed in which the protonation/deprotonation of the Mo(V) species is an essential step. Thus, one of the two protons involved in the catalytic cycle could be the one coupled to the molybdenum atom in the dissimilatory nitrate reductase of E. coli.

The molybdenum cofactor of Escherichia coli nitrate reductase A (NarGHI). Effect of a mobAB mutation and interactions with [Fe-S] clusters.

We have studied the effect of a mobAB mutation and tungstate on molybdo-molybdopterin-guanine dinucleotide (Mo-MGD) insertion into Escherichia coli nitrate reductase (NarGHI). Preparation of fluorescent oxidized derivatives of MGD (Form A and Form B) indicates that in a mobAB mutant there is essentially no detectable cofactor present in either the membrane-bound (NarGHI) or purified soluble (NarGH) forms of the enzyme. Electron paramagnetic resonance characterization of membrane-bound cofactor-deficient NarGHI suggests that it has altered electrochemistry with respect to the dithionite reducibility of the [Fe-S] clusters of NarH. Potentiometric titrations of membrane-bound NarGHI indicate that the NarH [Fe-S] clusters have midpoint potentials at pH 8.0 (Em,8.0 values) of +180 mV ([3Fe-4S] cluster), +130, -55, and -420 mV ([4Fe-4S] clusters) in a wild-type background and +180, +80, -35, and -420 mV in a mobAB mutant background. These data support the following conclusions: (i) a model for Mo-MGD biosynthesis and assembly into NarGHI in which both metal chelation and nucleotide addition to molybdopterin precede cofactor insertion; and (ii) the absence of Mo-MGD significantly affects Em,8.0 of the highest potential [4Fe-4S] cluster.

Further characterization of the two tetraheme cytochromes c3 from Desulfovibiro africanus: nucleotide sequences, EPR spectroscopy and biological activity.

The genes encoding the basic and acidic tetraheme cytochromes c3 from Desulfovibrio africanus have been sequenced. The corresponding amino acid sequences of the basic and acidic cytochromes c3 indicate that the mature proteins consist of a single polypeptide chain of 117 and 103 residues, respectively. Their molecular masses, 15102 and 13742 Da, respectively, determined by mass spectrometry, are in perfect agreement with those calculated from their amino acid sequences. Both D. africanus cytochromes c3 are synthesized as precursor proteins with signal peptides of 23 and 24 residues for the basic and acidic cytochromes, respectively. These cytochromes c3 exhibit the main structural features of the cytochrome c3 family and contain the 16 strictly conserved cysteine + histidine residues directly involved in the heme binding sites. The D. africanus acidic cytochrome c3 differs from all the other homologous cytochromes by its low content of basic residues and its distribution of charged residues in the amino acid sequence. The presence of four hemes per molecule was confirmed by EPR spectroscopy in both cytochromes c3. The g-value analysis suggests that in both cytochromes, the angle between imidazole planes of the axial histidine ligands is close to 90 degrees for one heme and much lower for the three others. Moreover, an unusually high exchange interaction (approximately 10[-2] cm[-1]) was evidenced between the highest potential heme (-90 mV) and one of the low potential hemes in the basic cytochrome c3. The reactivity of D. africanus cytochromes c3 with heterologous [NiFe] and [Fe] hydrogenases was investigated. Only the basic one interacts with the two types of hydrogenase to achieve efficient electron transfer, whereas the acidic cytochrome c3 exchanges electrons specifically with the basic cytochrome c3. The difference in the specificity of the two D. africanus cytochromes c3 has been correlated with their highly different content of basic and acidic residues.

Biochemical and spectroscopic characterization of two new cytochromes isolated from Desulfuromonas acetoxidans.

The multimeric cytochromes described to date in sulfate- and sulfur-reducing bacteria are associated with diverse respiratory modes involving the use of elemental sulfur or oxidized sulfur compounds as terminal acceptors. They exhibit no structural similarity with the other cytochrome c classes and are characterized by a bis-histidinyl axial iron coordination and low redox potentials. We have purified two new cytochromes c with markedly different molecular masses (10 000 and 50 000) from the bacterium Desulfuromonas acetoxidans, which uses anaerobic sulfur respiration as its sole energy source. The characterization by electrochemistry and optical and EPR spectroscopies revealed the cytochrome c (Mr = 10 000) to be the first monohemic cytochrome c exhibiting a bis-histidinyl axial coordination and a low redox potential (-220 mV). The cytochrome c (Mr = 50 000) contains four hemes of low potential (-200, -210, -370, and -380 mV) with the same axial coordination. The N-terminal amino acid sequences were compared with that of the trihemic cytochrome c7, previously described in D. acetoxidans and which is related to tetrahemic cytochrome c3 from sulfate reducing bacteria. Some homology was found between cytochrome c (Mr = 10 000) and cytochrome c7. Both D. acetoxidans cytochromes c are located in the periplasmic space and their biochemical and spectroscopic properties indicate that they belong to the class III cytochromes.

Nature and electronic structure of the Ni-X dinuclear center of Desulfovibrio gigas hydrogenase. Implications for the enzymatic mechanism.

The recent determination of the X-ray crystal structure of Desulfovibrio gigas hydrogenase has revealed that the active site is a Ni-X dinuclear center [Volbeda, A., Charon, M. H., Piras, C., Hatchikian, E. C., Frey, M., & Fontecilla-Camps, J. C. (1995) Nature 373, 580-587]. This unexpected result calls for a re-examination of the magnetic and redox properties that have been attributed previously to a mononuclear Ni center. We have used a combination of dosimetric and electron paramagnetic resonance (EPR) techniques to investigate the nature and the electronic structure of the Ni-X center in the redox forms of D. gigas hydrogenase giving EPR signals. The metal atom X was first shown to be Fe by accurate metal content analyses. Next, by determining the EPR characteristics of a polycrystal powder, it was shown that the redox form of the enzyme studied in the X-ray crystal experiments was essentially Ni-A. The temperature dependence of the Ni-A, Ni-B, Ni-C, and Ni-L EPR signals was studied over a large temperature range. No deviation from Curie's law could be detected, which places strong constraints upon the magnitude of the possible magnetic interactions between the Ni and Fe centers. When these results and the other available magnetic data are analyzed in the light of the crystal structure, it is concluded that the Fe center is diamagnetic in all the redox states of the enzyme. On the basis of these results, a mechanistic scheme consistent with a large body of experimental data can be proposed for Ni-containing hydrogenases.

Flavin mononucleotide-binding domain of the flavoprotein component of the sulfite reductase from Escherichia coli.

The flavoprotein component (SiR-FP) of the sulfite reductase from Escherichia coli is an octamer containing one FAD and one FMN as cofactors per polypeptide chain. We have constructed an expression vector containing the DNA fragment encoding for the FMN-binding domain of SiR-FP. The overexpressed protein (SiR-FP23) was purified as a partially flavin-depleted polymer. It could incorporate FMN exclusively upon flavin reconstitution to reach a maximum flavin content of 1.2 per polypeptide chain. Moreover, the protein could stabilize a neutral air-stable semiquinone radical over a wide range of pHs. During photoreduction, the flavin radical accumulated first, followed by the fully reduced state. The redox potentials, determined at room temperature [E'1 (FMNH./FMN) = -130 +/- 10 mV and E'2 (FMNH2/FMNH.) = -335 +/- 10 mV], were very close to those previously reported for Salmonella typhimurium SiR-FP [Ostrowski, J., Barber, M. J., Rueger, D. C., Miller, B. E., Siegel, L. M., & Kredich, N. M. (1989) J. Biol. Chem. 264, 15796-15808]. Both the radical and fully reduced forms of SiR-FP23 were able to transfer their electrons to cytochrome c quantitatively. Altogether, the results presented herein demonstrate that the N-terminal end of E. coli SiR-FP forms the FMN-binding domain. It folds independently, thus retaining the chemical properties of the bound FMN, and provides a good model of the FAD-depleted form of native SiR-FP. Moreover, the FMN prosthetic group in SiR-FP23 and native SiR-FP is compared to that of cytochrome P450 reductase and bacterial cytochrome P450, which also contain one FAD and one FMN per polypeptide chain.