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An alternative route for haem b biosynthesis is operative in sulfate-reducing bacteria of the Desulfovibrio genus and in methanogenic Archaea. This pathway diverges from the canonical one at the level of uroporphyrinogen III and progresses via a distinct branch, where sirohaem acts as an intermediate precursor being converted into haem b by a set of novel enzymes, named the alternative haem biosynthetic proteins (Ahb). In this work, we report the biochemical characterisation of the Desulfovibrio vulgaris AhbD enzyme that catalyses the last step of the pathway. Mass spectrometry analysis showed that AhbD promotes the cleavage of S -adenosylmethionine (SAM) and converts iron-coproporphyrin III via two oxidative decarboxylations to yield haem b , methionine and the 5′-deoxyadenosyl radical. Electron paramagnetic resonance spectroscopy studies demonstrated that AhbD contains two [4Fe–4S] 2 +/1 + centres and that binding of the substrates S -adenosylmethionine and iron-coproporphyrin III induces conformational modifications in both centres. Amino acid sequence comparisons indicated that D. vulgaris AhbD belongs to the radical SAM protein superfamily, with a GGE-like motif and two cysteine-rich sequences typical for ligation of SAM molecules and iron-sulfur clusters, respectively. A structural model of D. vulgaris AhbD with putative binding pockets for the iron-sulfur centres and the substrates SAM and iron-coproporphyrin III is discussed.

The effect of amino acid residues modification of Desulfovibrio gigas hydrogenase on different activity assays is reported. The first method consisted in the modification of glutamic and aspartic acid residues of the enzyme with ethylenediamine in order to change the polarity of certain regions of the protein surface. The second method consisted in the modification of histidine residues with a Ru complex in order to change the acid-base properties of the histidine residues. The implication of these modifications in the enzyme kinetics has been studied by measuring in parallel the activities of para/ortho hydrogen conversion, deuterium/hydrogen exchange and dyes reduction with hydrogen. Our experimental data support some hypothesis based on the three-dimensional structure of this enzyme: (a) electrostactic interactions between the hydrogenase and the redox partner play an essential role in the kinetics; (b) the histidine ligand and the surrounding acidic residues of the distal [4Fe4S] cluster form the recognition site of the redox partner of the hydrogenase; and (c) histidine residues are involved in the hydron transfer pathway of the hydrogenase.

A cytochrome c nitrite reductase (NiR) was purified for the first time from a microorganism not capable of growing on nitrate, the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough. It was isolated from the membranes as a large heterooligomeric complex of 760 kDa, containing two cytochrome c subunits of 56 and 18 kDa. This complex has nitrite and sulfite reductase activities of 685 micromol NH(4)(+)/min/mg and 1.0 micromol H(2)/min/mg. The enzyme was studied by UV-visible and electron paramagnetic resonance (EPR) spectroscopies. The overall redox behavior was determined through a visible redox titration. The data were analyzed with a set of four redox transitions, with an E(0)' of +160 mV (12% of total absorption), -5 mV (38% of total absorption), -110 mV (38% of total absorption) and -210 mV (12% of total absorption) at pH 7.6. The EPR spectra of oxidized and partially reduced NiR show a complex pattern, indicative of multiple heme-heme magnetic interactions. It was found that D. vulgaris Hildenborough is not capable of using nitrite as a terminal electron acceptor. These results indicate that in this organism the NiR is not involved in the dissimilative reduction of nitrite, as is the case with the other similar enzymes isolated so far. The possible role of this enzyme in the detoxification of nitrite and/or in the reduction of sulfite is discussed.

Flavodoxins are proteins with an α/β doubly wound topology that mediate electron transfer through a non-covalently bound flavin mononucleotide (FMN). The FMN moiety binds strongly to folded flavodoxin ( K D =0.1 nM, oxidized FMN). To study the effect of this organic cofactor on the conformational stability, we have characterized apo and holo forms of Desulfovibrio desulfuricans flavodoxin by GuHCl-induced denaturation. The unfolding reactions for both holo- and apo-flavodoxin are reversible. However, the unfolding curves monitored by far-UV circular dichroism and fluorescence spectroscopy do not coincide. For both apo- and holo-flavodoxin, a native-like intermediate (with altered tryptophan fluorescence but secondary structure as the folded form) is present at low GuHCl concentrations. There is no effect on the flavodoxin stability imposed by the presence of the FMN cofactor (Δ G =20(±2) and 19(±1) kJ/mol for holo- and apo-flavodoxin, respectively). A thermodynamic cycle, connecting FMN binding to folded and unfolded flavodoxin with the unfolding free energies for apo- and holo-flavodoxin, suggests that the binding strength of FMN to unfolded flavodoxin must be very high ( K D =0.2 nM). In agreement, we discovered that the FMN remains coordinated to the polypeptide upon unfolding.

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.

Three c -type cytochromes were isolated and characterized from the sulfate reducer Desulfovibrio africanus . A basic tetraheme cytochrome c 3 of molecular mass 16 kDa was previously described and we have extended its characterization. Two other c 3 -type cytochromes, not previously observed, have also been characterized. These include an acidic tetraheme cytochrome c 3 of molecular mass 15 kDa and an octaheme dimeric cytochrome c 3 with a native size of 35 kDa. This is the first report of the presence of two distinct tetraheme cytochromes c 3 in a Desulfovibrio species. The physico-chemical properties of the three cytochromes, including optical properties, iron content, cysteine and histidine content, N-terminal amino sequence and redox properties, are characteristic of cytochrome c 3 family. The acidic tetraheme cytochrome c 3 exhibited similar midpoint potential values for all four hemes ( E m1 = −210 mV; E m2 = −240 mV E m3 = −260 mV E m4 = −270 mV) whereas in the basic tetraheme cytochrome c 3 one heme had a much more positive potential than the others ( E m1 = −90 mV E m2 = −260 mV E m3 = −280 mV E m4 = −290 mV The acidic tetraheme cytochrome c 3 exhibited unique properties including amino-acid composition and poor reactivity towards hydrogenase. However, it is readily reduced by this enzyme in the presence of the basic cytochrome c 3 . The weak reactivity of the acidic tetraheme cytochrome c 3 towards hydrogenase has been correlated with its low content of basic residues.

Resonance Raman spectra of the molybdenum containing aldehyde oxidoreductase from Desulfovibrio gigas were recorded at liquid nitrogen temperature with various excitation wavelengths. The spectra indicate that all the iron atoms are organised in [2Fe2S] type centers consistent with cysteine ligations. No vibrational modes involving molybdenum could be clearly identified. The features between 280 and 420 cm−1 are similar but different from those of typical plant ferredoxin-like [2Fe2S] cluster. The data are consistent with the presence of a plant ferredoxin-like cluster (center I) and a unique [2Fe2S] cluster (center II), as suggested by other spectroscopic studies. The Raman features of center II are different from those of other [2Fe2S] clusters in proteins. In addition, a strong peak at ca. 683 cm−, which is not present in other [2Fe2S] clusters in proteins, was observed with purple excitation (406.7-413.1 run). The peak is assigned to enhanced cysteinyl CS stretching in center II, suggesting a novel geometry for this center.

Desulfovibrio africanus ferredoxin I was studied by magnetic circular dichroism and 1 H-NMR spectroscopies. These showed the presence of histidine and tryptophan, in contrast to the previously reported amino-acid sequence (Bruschi and Hatchikian (1982) Biochimie 64, 503–507). This was redetermined and the revised sequence shown to contain both histidine and tryptophan, as well as four other corrections (Sery et al. (1994) Biochemistry, submitted). Electrospray mass spectrometry confirmed the mass of the ferredoxin was that given by the revised amino-acid sequence. The secondary structure of the ferredoxin I was investigated with two-dimensional 1 H-NMR experiments and both α-helix and β-sheet structure detected. The influence of the paramagnetism of the Fe 4 S 4 cluster on the NMR properties of the ferrredoxin protons was investigated, by temperature-dependent experiments, and it was concluded that there is only a negligible dipolar contribution to resonance chemical shifts from this source. The significance of this for the determination of the three-dimensional structure of the ferredoxin by NMR is discussed.

The amino-acid sequence of an octaheme cytochrome c 3 isolated from Desulfovibrio desulfuricans Norway is presented. The protein molecule ( M r 26 000) comprises two identical subunits of 111 amino acids with the characteristics typical of tetrahemic cytochrome c 3 class. Comparisons between the amino-acid sequences and physiological properties of cytochrome c 3 ( M r 26 000) and cytochromes c 3 ( M r 13 000) isolated from various species of Desulfovibrio showed the existence of considerable differences. In order to distinguish between the various subclasses in the cytochrome c 3 superfamily, the amino-acid sequence of cytochrome c 3 ( M r 26000 ) was compared with six known cytochrome c 3 ( M r 13000 ) sequences as well as with the sequence of the four c 3 -like domains of a high molecular weight cytochrome c (Hmc) containing 16 hemes per molecule of 65 500 Da, isolated from Desulfovibrio vulgaris Hildenborough. The evolution and phylogenetic relationships of these various polyhemic cytochromes are discussed.

The amino-acid sequence of the cytochrome c-553 from Desulfovibrio desulfuricans Norway has been determined and compared with that of two different cytochromes c-553 from D. vulgaris already described and with that cytochrome c-551 from Pseudomonas. This low-molecular-weight monohemic cytochrome comprises 80 amino acids and has the typical characteristics of small cytochromes such as mitochondrial cytochromes. Secondary-structure predictions are deduced from sequence data and are compared with X-ray three-dimensional structures of low-molecular-weight cytochrome c. The phylogenetic situation of Desulfovibrio cytochromes c-533 in the cytochrome c superfamily is discussed.

The gene encoding a protein containing a novel iron sulfur cluster ([6Fe-6S]) has been cloned from Desulfovibrio desulfuricans ATCC 27774 and sequenced. An open reading frame was found encoding a 545 amino acid protein ( M r 58 496). The amino acid sequence is highly homologous with that of the corresponding protein from D. vulgaris (Hildenborough) and contains a Cys-motif that may be involved in coordination of the Fe-S cluster.

The structural genes coding for the flavodoxin proteins from two different strains of the sulfate-reducing bacteria Desulfovibrio gigas (ATCC 19364/NCIB 9332 and ATCC 29494/DSM 496) have been identified, cloned and the nucleotide sequence established. The protein sequences derived from the gene from each strain share a sequence identity of 66% with regions directly involved in binding the flavin mononucleotide cofactor being the most homologous. Both aromatic residues that flank the flavin isoalloxazine ring in the crystal structure of the flavodoxin from D. vulgaris, i.e., Trp-60 and Tyr-98, are also present in these flavodoxin proteins. These observations stand in contrast to reports that the flavodoxin from Desulfovibrio gigas contains a single tryptophan residue which is located distant from the flavin binding site. Therefore, the FMN binding site of this flavodoxin is not distinct from the other Desulfovibrio flavodoxins in this regard.

We have obtained direct evidence that the majority of the sirohydrochlorin chromophore in the dissimilatory sulfite reductase desulfoviridin from Desulfovibrio gigas, is not associated with any metal. The evidence comes from resonance Raman measurements of native and deuterated samples of desulfoviridin. The breathing mode v4 (or v4*) at 1336 cm-1 in the native enzyme is downshifted to 1326 cm-1 upon deuteration. This mode is not sensitive to deuteration if a metal is present at the center of the chromophore inside protein or in solution. The results also establish the existence of exchangeable core hydrogen(s) at the pyrrolic nitrogen(s).

The gene coding for the flavodoxin protein from Desulfovibrio desulfuricans [Essex 6] (ATCC 29577) has been cloned and sequenced. The gene was identified on Southern blots of HindIII-digested genomic DNA by hybridization to the coding region for the flavodoxin from Desulfovibrio vulgaris [Hildenborough] (Krey, G.D., Vanin, E.F. and Swenson, R.P. (1988) J. Biol. Chem. 263, 15436-15443). Ultimately, a 1.8 kb TaqI fragment was cloned which contains an open reading frame of 447 nucleotides coding for an acidic protein of 148 amino acids and calculated molecular weight of 15,726. The derived amino acid sequence of this protein is 47% identical to the flavodoxin from D. vulgaris. Regions of the polypeptide which form the flavin mononucleotide binding site are largely homologous; however, some perhaps significant differences are noted. The aromatic amino acid residues that flank the flavin isoalloxazine ring in the D. vulgaris structure, i.e., tryptophan-60 and tyrosine-98, are conserved in this flavodoxin.

1. 1. Cell-free extracts from Desulfovibrio desulfuricans reduce hydroxylamine to ammonia at the expense of molecular hydrogen by a non-specific process in which hydrogenase is the only enzymic factor. The mechanism consists of the reduction by hydrogenase of a natural electron carrier, cytochrome c 3 , which is chemically reoxidized by hydroxylamine without the intervention of a specific reductase. 2. 2. Benzyl-viologen can be substituted for the cytochrome and ensure the chemical coupling of hydrogenase activity with hydroxylamine reduction. However, the efficiency of this artificial carrier is less than that of cytochrome c 3 . 3. 3. Purified ferro-cytochrome c from beef heart is, like cytochrome c 3 , chemically reoxidized by hydroxylamine, but its rate of reoxidation is lower. 4. 4. The kinetics of hydroxylamine reduction by the extracts have been studied as a function of temperature, substrate concentration, and carrier concentration (both natural and artificial). 5. 5. Experiments which have been performed with other anaerobic and aerobic bacteria provide some evidence that the reduction of hydroxylamine by hydrogenase and a non-specific natural or artificial carrier is not confined to the sulphate-reducing bacteria but can be effected by any organism possessing hydrogenase.

Hydrogenase from Desulfovibrio desulfuricans has been purified about fifty-fold. At the highest level of purity 1 mg of protein N catalyses the oxidation of 2.6·10 6 μl of H 2 per hour by methylene blue at 34°. Purified hydrogenase was found to be activated by FeCl 2 or FeCl 3 , the increase in activity being the same with benzyl viologen or methylene blue as oxidant. A thermostable cytochrome-like component was obtained during the purification, which has no hydrogenase activity, but could be reduced by hydrogen and hydrogenase and reoxidised by O 2 , methylene blue or FMN. Methylene blue and benzyl viologen were reduced rapidly and at the same rate by hydrogen in the presence of purified hydrogenase; FMN, FAD and riboflavin were reduced at about one-hundredth of this rate, while DPN, TPN, sulfate and ferricyanide were not reduced. The optimum pH for methylene blue reduction was 8.4, cysteine or glutathione was essential for enzyme activity and serum albumin had a protective action at high enzyme dilutions. Hydrogenase was inhibited by p -chloromercuribenzoate and the inhibition was reversed by cysteine or glutathione. Cyanide and other metal-binding agents, hydroxylamine and nitrite were also inhibitory.

Ferredoxin II from Desulphovibrio gigas is a tetrameric protein containing a novel iron-sulphur cluster consisting of three iron atoms. The low-temperature magnetic circular dichroism (MCD) spectra of the oxidized and dithionite-reduced forms of ferredoxin II have been measured over the wavelength range approx. 300-800 nm. Both oxidation levels of the cluster are shown to be paramagnetic, although only the oxidized form gives an EPR signal. MCD magnetization curves have been constructed over the temperature range approx. 1.5-150 K and at fields between 0 and 5.1 Tesla. The curve for the oxidized protein can be fitted to a ground state of spin S = 1/2 with an isotropic g factor of 2.01. There is evidence for the thermal population of a low-lying electronic state above 50 K. The reduced protein gives a distinctive set of magnetization curves that are tentatively assigned to a ground state of S = 2, with a predominantly axial zero-field distortion that leaves the doublet Ms = +/-2 lowest in energy. The zero-field components have a maximum energy spread of approx. 15 cm-1. which places an upper limit of 4 cm-1 on the axial zero-field parameter D. The MCD spectra of the oxidized and reduced forms of the cluster are quite distinctive from one another. The spectra of the oxidized state are also different from those of oxidized high-potential iron protein from Chromatium and should provide a useful criterion for distinguishing between four- and three-iron clusters in their highest oxidation levels.

The coordination geometry at the heme iron of the cytochromes c-553 from Desulfovibrio vulgaris and Desulfovibrio desulfuricans was investigated by 1H-nuclear magnetic resonance and circular dichroism spectroscopy. Individual assignments were obtained for heme c and the axial ligands. From studies of nuclear Overhauser enhancements the axial histidine imidazole ring orientation relative to the heme group was found to coincide with other c-type cytochromes. In contrast, a new structure was observed for the axial methionine in the reduced cytochromes c-553. This includes S chirality at the iron-bound sulfur atom, but compared to cytochromes c-551 from Pseudomonads and Rhodopseudomonas gelatinosa and cytochrome c5 from Pseudomonas mendocina, which also contain S-chiral methionine, a different spatial arrangement of the gamma- and beta-methylene groups and the alpha carbon of methionine prevails. For the ferricytochromes c-553 R chirality was found for the iron-bound sulfur. This is the first observation of different methionine chirality in different oxidation states of the same c-type cytochrome.

A flavodoxin and a rubredoxin have been isolated from the sulfate-reducing bacterium Desulphovibrio salexigens (strain British Guiana, NICB 8403). Their amino acid composition and spectral characteristics did not differ markedly from the homologous proteins presented in other Desulphovibrio spp. Flavodoxin was shown to be active in the electron transport of the sulfite reductase system.

Cytochrome c-553 of Desulfovibrio vulgaris, Miyazaki, was purified to homogeneity. The absorption spectrum of the ferro form has four peaks at 553, 525, 417 and 317 nm with a plateau near 280 nm, and that of the ferri form has three peaks at 525, 410 and 360 nm with a plateau near 280 nm and a shoulder at 560 nm. The millimolar absorbance coefficient of the alpha-peak of the ferro form is 23.9. The molecular weight of cytochrome c-553 is 8000, and it contains one heme. Its isoelectric point is rather alkaline, and its standard redox potential is -0.26 V at pH 7.0. Its amino acid composition is unique; it lacks proline, isoleucine and tryptophan. Ferrocytochrome c-553 does not combine with CO, nor does it transfer electrons directly to various redox carriers such as flavin nucleotides, methylene blue, indigodisulfonate, 5-methylphenazinium methyl sulfate, 1-methoxy-5-methylphenazinium methyl sulfate, viologens and cytochrome c3, but is oxidized by ferricyanide or by O2. Cytochrome c-553 can be reduced by formate dehydrogenase of this bacterium in the presence of formate, but not by hydrogenase under H2. The formate dehydrogenase does not reduce cytochrome c3 in the presence of formate. The systematic name for formate dehydrogenase of D. vulgaris is, therefore, established as formate:ferricytochrome c-553 oxidoreductase in EC subclass 1.22.-.

(1) Reduction process of cytochrome c 3 by hydrogenase (EC 1.12.2.1) under H 2 was analyzed by means of spectrophotometry. (2) When cytochrome c 3 is in equilibrium with H 2 under reduced pressure, spectral abnormality in the Soret region appeared most significantly at 1 4 reduction state, less significantly at 1 2 reduction state, and disappeared at 2 4 reduction state. (3) The spectral changes during the enzymic reduction of cytochrome c 3 in H 2 -saturated solution traced at several wavelengths also showed spectral abnormality in the Soret region at the early stage of reaction. (4) The first-order rate constants for the successive reduction steps from all-ferric to all-ferrous form of cytochrome c 3 was estimated to be k 1 = 0.061s −1 , k 2 = 0.063s −1 , k 3 = 0.039s −1 and k 4 = 0.014s −1 (cytochrome c 3 : 2 μM; hydrogenase: 2 nM, and at 20°C, pH 7.0). (5) Strong interaction is suggested between hemes 3 and 4 (for the refined structure and heme-numbering, see Higuchi, Y., Kusunoki, M., Matsuura, Y., Yasuoka, N. and Kakudo, M. (1984) J. Mol. Biol. 172, 109–139). (6) The first electron from hydrogenase is supposed to be transferred to these hemes and delocalized between them, and the second electron, among hemes 3, 4 and 1. (7) The characteristic behavior in the enzymic reduction of cytochrome c 3 is different from that in non-enzymic reduction.

1. Cytochrome c 3 , a unique hemoprotein with a negative redox potential and four heme groups bound to a single polypeptide chain, reacts with imidazole in the reduced state to form a low-spin ferro · imidazole complex which is spectrally characterized by a 3.1 nm blue shift in the α-peak (from 550.5 to 547.4 nm). The spectral imidazole · cytochrome c 3 complex is detectable at 77 but not at 298 K. 2. Mammalian ferrocytochrome c did not undergo a spectral interaction with imidazole at either 77 or 298 K, indicating that the imidazole · cytochrome c 3 complex reflects a unique event for cytochrome c 3 . 3. Formation of the imidazole · cytochrome c 3 complex is strongly dependent on imidazole concentration (apparent K d of approx. 50 mM), and is abolished in the presence of 100 mM phosphate. This latter effect is attributable to formation of an imidazole · phosphate complex. A pH titration of the imidazole · cytochrome c 3 spectral complex implicates ionization of an imidazole function (p K = 8.5). 4. EPR studies at 8.5 K of ferricytochrome c 3 before and after one reduction-oxidation cycle indicate that at least two of the hemes undergo reaction with imidazole forming two different low-spin ferric heme · imidazole complexes, with significant shifts in the g values of two heme signals. 5. The spectral and EPR results are consistent with formation as the primary event of a low-spin ferrocytochrome c 3 · imidazole complex in which increased hydrophobicity and protonation-deprotonation effects are contributary to the consequent lability of cytochrome c 3 .

Rubredoxin and two distinct ferredoxins have been purified from Desulfovibrio africanus. The rubredoxin has a molecular weight of 6000 while the ferredoxins appear to be dimers of identical subunits of approximately 6000 to 7000 molecular weight. Rubredoxin contains one iron atom, no acid-labile sulfide and four cysteine residues per molecule. Its absorbance ratio A 278 A 490 is 2.23 and its amino acid composition is characterized by the absence of leucine and a preponderance of acidic amino acids. The two ferredoxins, designated I and II, are readily separated on DEAE-cellulose. The amino acid compositions of ferredoxins I and II show them to be different protein species; the greater number of acidic amino acid residues in ferredoxin I than in ferredoxin II appears to account for separation based on electronic charge. Both ferredoxins contain four iron atoms, four acid-labile sulfur groups and either four (ferredoxin II) or six (ferredoxin I) cysteine residues per molecule. Spectra of the two ferredoxins differ from those of ferredoxins of other Desulfovibrio species by exhibiting a pronounced absorption peak at 283 nm consistent with an unusual high content of aromatic residues. The A 385 A 283 absorbance ratio of ferredoxins I and II are 0.56 and 0.62, respectively. The N-terminal sequencing data of the two ferredoxins clearly indicate that ferredoxins I and II are different protein species. However, the two proteins exhibit a high degree of homology. The physiological activity of ferredoxins I and II appears to be similar as far as the electron transfer in the phosphoroclastic reaction is concerned.

Reductive titration curves of flavodoxin from Desulfovibrio vulgaris displayed two one-electron steps. The redox potential E-2 for the couple oxidized flavodoxin/flavodoxin semiquinone was determined by direct titration with dithionite. E-2 was -149 plus or minus 3 mV (pH 7.78, 25 degrees C). The redox potential E-1 for the couple flavodoxin semiquinone/fully reduced flavodoxin was deduced from the equilibrium concentration of these species in the presence of hydrogenase and H-2. E-1 was -438 plus or minus 8 mV (pH 7.78, 25 degrees C). Light-absorption and fluorescence spectra of flavodoxin in its three redox states have been recorded. Both the rate and extent of reduction of flavodoxin semiguinone with dithionite were found to depend on pH. An equilibrium between the semiquinone and hydroquinone forms occurred at pH values close to the neutrality, even in the presence of a large excess of dithionite, suggesting an ionization in fully reduced flavodoxin with a pK-a = 6.6. The association constants K for the three FMN redox forms with the apoprotein were deduced from the value of K (K = 8 times 10-7 M-1) measured with oxidized EMN at pH 7.0. Oxidized flavodoxin was found to comproportionate with the fully reduced protein (k-comp = 4.3 times 10-3 M-1 times s-1, pH 9.0, 22 degrees C) and with reduced free FMN (K-comp = 44 M-1 times s-1, pH 8.1, 20 degrees C). Fast oxidation of reduced flavodoxin occurred in the presence of O-2. Slower oxidation of semiquinone was dependent on pH in a drastic way.

Summary Two ferredoxins isolated from Desulfovibrio desulfuricans Norway have been purified and characterized. The less acidic, designated as ferredoxin I, contains four iron atoms, four acid-labile sulfur groups and six cysteine residues per molecule. Ferredoxin II is more acidic and abundant than ferredoxin I, but is very unstable to O 2 . Ferredoxin I and ferredoxin II differ according to amino acid composition but are homologous with respect to their N-terminal amino acid sequence. The absorption spectra of the two ferredoxins are similar to those of other Desulfovibrio species. Both proteins appear to be dimers of identical 6000-dalton subunits. Their activity was tested in two types of reaction in the electron transfer chain (phosphoroclastic reaction and sulfite reductase activity). The isolation of two different ferredoxins from the same organism, Desulfovibrio , has been reported in Desulfovibrio africanus but the significance of two ferredoxins functioning in the same electron transfer chain is not yet understood.

Desulphoviridin in the oxidized state showed EPR signals around g = 6, consistent with the sirohaem being in the high-spin ferric state. This was unreactive with sulphite, sulphide or cyanide; but readily reduced by methyl viologen. When the enzyme was treated with Na 2 S 2 O 4 the sirohaem was slowly reduced and a spectrum of a reduced iron-sulphur cluster at g = 2.07, 1.93, 1.91 appeared over the course of an hour. An intermediate in this reaction was indicated by a free radical signal which appeared within seconds and then gradually disappeared. On treatment with nitrite and reduced methyl viologen, the enzyme gave a spectrum of a nitroxide derivative similar to that seen with plant nitrite reductase. The midpoint reduction potential of the haem was estimated to be −310 mV or less. The iron-sulphur cluster has a very low potential, being only reduced in the presence of free Na 2 S 2 O 4 around −560 mV. Desulphoviridin can be classed with sirohaem-containing iron-sulphur proteins.

The ultraviolet visible, and near infrared spectrum of a two-iron protein from Desulphovibrio gigas, a new type of non-haem iron protein lacking labile sulphide, is compared with that of D. gigas rubredoxin. The charge transfer band maxima of rubredoxin at 495 and 565 nm are less separated in the new protein implying a higher symmetry of the two iron centres. The existence of a spin-spin interaction between the two iron centres in the new protein is suggested by the magnetic susceptibility measurements of the oxidized and reduced states of both proteins, which gives a smaller value per iron centre for the new protein. The oxidized form of the two iron-protein has a complex EPR spectrum with signals at g values of 8.97, 7.72, 5.73, 4.94 and 1.84. An EPR titration gives a value of −35 ± 15 mV for the two signals at g values of 7.72 and 5.73. Rubredoxin has the characteristic spectrum of rubredoxins with two signals at g values of 9.4 and 4.27.

The isolation, purification, and partial characterization of a novel iron-containing protein from the sulfate-reducing anaerobic bacterium, Desulfovibrio gigas , is described. The highly insoluble protein was isolated from the cell debris following osmotic shock of the bacteria. The insoluble fraction consistently contained about 90% of the cell-associated iron. Elemental analysis of a crude protein preparation gave 5.3% iron, 2.9% sulfur and 11.9% nitrogen. An independent colorimetric iron analysis showed 6.4% iron. The iron could be dissociated from the protein by treatment with 5% SDS. The iron-free protein was purified by a combination of organic extraction and DEAE-cellulose chromatography. The purified protein showed only one major band, M r 14 000, by SDS-polyacrylamide gel electrophoresis. The protein could be reconstituted upon treatment with an appropriate mixture of FeS and β-mercaptoethanol. The reconstituted protein had the same physical and chemical properties as the native protein. The amino acid composition was not unusual except for the high isoleucine content.

The type and the amount of end products resulting from sulfite reduction catalysed by a single partially purified desulfoviridin preparation from Desulfovibrio gigas were shown to depend upon the enzymic assay conditions employed. Both manometric and spectrophotometric assays were used, with reduced methyl viologen serving as the electron donor in each system. Trithionate, thiosulfate, tetrathionate and sulfide were identified as possible end products. In the manomeric assays, sulfide production was favoured by high reduced methyl viologen concentrations, low sulfite concentrations and a pH value of 7.0 as opposed to 6.0. In the spectrophotometric assays, results approaching the stoichiometric conversion of sulfite to sulfide were obtained only at high initial reduced methyl viologen concentrations.

(1) Cytochromes c3 from Desulfovibrio vulgaris (Hildenborough strain) and Desulfovibrio desulfuricans (Norway strain) have been studied by differential pulse polarography and spectroelectrochemical method. Both cytochromes exhibit two reduction potential values Eh approximately --0.25 and --0.34 V (D. vulgaris), Eh approximately --0.16 and --0.34 V (D. desulfuricans). (2) Titrations by dithionite and controlled potential electrolysis coupled with polarography and spectrophotometry suggest that in both cases electronic exchanges are rapid.

The interaction of various exogenous ligands (nitrite, hydroxylamine, cyanide, azide, imidazole, fluoride, chloride, ammonia, histidine or Tris) on the oxidation-reduction states of cytochrome c 3 has been studied by low-temperature EPR. The reaction of NO or hydroxylamine with cytochrome c 3 results in the first instance of nitrogen-liganded hyperfine interaction ( 14 N hyperfine splitting: 15.46) in an intact cytochrome. Weak interactions, if any, are noted in the ferric state. Varying degrees of interaction with a paramagnetic half-reoxidized intermediate are indicated. Reoxidation, especially in the presence of NO, cyanide, fluoride or imidazole, results in altered and labile ferric heme systems which then undergo changes in EPR absorption. Primary amino-containing compounds apparently react to form a specific cytochrome c 3 -product intermediate at half-reoxidation. The results obtained appear consistent with a stacked heme configuration in the ferric state which undergoes a finite change on reduction to accomodate ligand interaction.

1. 1. EPR and optical studies provide for differentiation of the c-type cytochromes examined into three groups with different heme contents. The three groups consist of: the mono-heme type, cytochrome c-553; cytochromes c3 and c′3 containing at least two hemes per molecule; and cytochromes cc3 and cc′3 which contain at least four hemes per molecule. This division into three groups agrees with a previous similar grouping based on amino acid composition and/or sequence. 2. 2. EPR studies suggest that heme-heme interaction is manifest in the ferric state of cytochrome c3. At about a half-reduced state, an EPR-detectable intermediate with a decreased degree of interaction between hemes is observed. 3. 3. Based on EPR and light absorption changes, cytochrome c3 is extremely stable in 8 M urea in the ferric state. However, repeated reduction and reoxidation in the presence of 8 M urea results in the apparent conversion of the multi-heme system to a mono-heme system. 4. 4. These and other observations suggest a conformation change involving a possible reorientation of the multi-heme moieties in the partially or fully-reduced tate. Depending on the reactant(s) present this process may be reversible.

1. The H2:fumarate reductase activities of particulate fractions prepared from Desulfovibrio gigas grown on various media (lactate-sulfate, fumarate or fumarate-sulfate) were measured. Activity was higher in the particulate fraction prepared from cells grown on fumarate or fumarate-sulfate media than in those prepared from cells cultivated on lactate-sulfate. 2. Various inhibitors were tested for their effect on fumarate reduction by H2 in a particulate fraction of Desulfovibrio gigas. A strong inhibitory effect was observed with 2-hetyl-4-hydroxyquinoline-N-oxide and antimycin A. These compounds act as inhibitors of an intermediary electron carrier between hydrogenase and fumarate reductase. 3. The strict anaerobe Desulfovibrio gigas has been found to contain a b-type cytochrome linked to the particulate H2:fumarate reductase activity. A protoheme obtained by extraction from the particulate fraction was characterized by its reduced pyridine hemochromogen.

Cytochrome cc′3, flavodoxin and ferredoxin are able to stimulate the reduction of thiosulfate by molecular H2 in an extract of the sulfate reducer Desulfovibrio gigas. Only flavodoxin and ferredoxin will stimulate the reduction of sulfite by the same extract, whereas cytochrome cc′3 is specific for thiosulfate reduction. Sulfite accumulates during the early stage of thiosulfate reduction and is reduced only when thiosulfate has disappeared. The results are discussed in terms of the regulation, by these different electron carriers, of the electron flow from H2 to the various sulfur compounds acting as terminal electron acceptors in sulfate-reducing bacteria.

Cytochromes c with absorption spectra and oxidation-reduction potentials very similar to those of Desulfovibrio vulgaris cytochrome c3 were isolated from the photosynthetic bacteria Chloropseudomonas ethylicum, Rhodopseudomonas spheroides and Rps. palustris, as well as from the blue-green alga Anacystis nidulans. Analysis of the highly purified C. ethylicum cytochrome c-551.5 indicates that this protein contains three heme groups per molecule, as compared to Desulfovibrio cytochrome c3, which has four hemes. It is suggested that a special class of cytochromes c be recognized which has absorption spectra and oxidation-reduction potentials similar to those of Desulfovibrio cytochrome c3.

An assay procedure for adenosine 5′-phosphosulfate reductase from Desulfovibrio desulfuricans and Thiobacillus thioparus is described that utilizes the AMP-dependent reduction of ferricyanide occurring in the presence of SO32−, and optimum conditions for the assay are reported. The stoichiometry of this oxidation was determined and indicates that the reaction proceeds as follows: AMP + SO32− + 2Fe(CN)63− → adenosine 5′-phosphosulfate + 2Fe(CN)64−. Other nucleotides, deAMP, IMP, and GMP, can replace AMP in the reaction. Employing this assay, adenosine 5′-phos phosulfate reductase was purified from extracts of D. desulfuricans, and the purified enzyme, essentially homogenous in the ultracentrifuge, contains flavin adenine dinucleotide and iron. Inhibition of the enzyme by p-hydroxymercuribenzoate and reversal of the inhibition by monothiols indicate that one or more sulfhydryl groups are required for activity.