Comparative Fe and Zn K-edge X-ray absorption spectroscopic study of the ferroxidase centres of human H-chain ferritin and bacterioferritin from Desulfovibrio desulfuricans.

Iron uptake by the ubiquitous iron-storage protein ferritin involves the oxidation of two Fe(II) ions located at the highly conserved dinuclear "ferroxidase centre" in individual subunits. We have measured X-ray absorption spectra of four mutants (K86Q, K86Q/E27D, K86Q/E107D, and K86Q/E27D/E107D, involving variations of Glu to Asp on either or both sides of the dinuclear ferroxidase site) of recombinant human H-chain ferritin (rHuHF) in their complexes with reactive Fe(II) and redox-inactive Zn(II). The results for Fe-rHuHf are compared with those for recombinant Desulfovibrio desulfuricans bacterioferritin (DdBfr) in three states: oxidised, reduced, and oxidised/Chelex-treated. The X-ray absorption near-edge region of the spectrum allows the oxidation state of the iron ions to be assessed. Extended X-ray absorption fine structure simulations have yielded accurate geometric information that represents an important refinement of the crystal structure of DdBfr; most metal-ligand bonds are shortened and there is a decrease in ionic radius going from the Fe(II) to the Fe(III) state. The Chelex-treated sample is found to be partly mineralised, giving an indication of the state of iron in the cycled-oxidised (reduced, then oxidised) form of DdBfr, where the crystal structure shows the dinuclear site to be only half occupied. In the case of rHuHF the complexes with Zn(II) reveal a surprising similarity between the variants, indicating that the rHuHf dinuclear site is rigid. In spite of this, the rHuHf complexes with Fe(II) show a variation in reactivity that is reflected in the iron oxidation states and coordination geometries.

Effect of hydrogen-bond networks in controlling reduction potentials in Desulfovibrio vulgaris (Hildenborough) cytochrome C3 probed by site-specific mutagenesis.

Cytochromes C3 isolated from Desulfovibrio spp. are periplasmic proteins that play a central role in energy transduction by coupling the transfer of electrons and protons from hydrogenase. Comparison between the oxidized and reduced structures of cytochrome C3 isolated from Desulfovibrio vulgaris (Hildenborough) show that the residue threonine 24, located in the vicinity of heme III, reorients between these two states [Messias, A. C., Kastrau, D. H. W., Costa, H. S., LeGall, J., Turner, D. L., Santos, H., and Xavier, A. V. (1998) J. Mol. Biol. 281, 719-739]. Threonine 24 was replaced with valine by site-directed mutagenesis to elucidate its effect on the redox properties of the protein. The NMR spectra of the mutated protein are very similar to those of the wild type, showing that the general folding and heme core architecture are not affected by the mutation. However, thermodynamic analysis of the mutated cytochrome reveals a large alteration in the microscopic reduction potential of heme III (75 and 106 mV for the protonated forms of the fully reduced and oxidized states, respectively). The redox interactions involving this heme are also modified, while the remaining heme-heme interactions and the redox-Bohr interactions are less strongly affected. Hence, the order of oxidation of the hemes in the mutated cytochrome is different from that in the wild type, and it has a higher overall affinity for electrons. This is consistent with the replacement of threonine 24 by valine preventing the formation of a network of hydrogen bonds, which stabilizes the oxidized state. The mutated protein is unable to perform a concerted two-electron step between the intermediate oxidation stages, 1 and 3, which can occur in the wild-type protein. Thus, replacing a single residue unbalances the global network of cooperativities tuned to control thermodynamically the directionality of the stepwise electron transfer and may affect the functionality of the protein.

The mechanism of superoxide scavenging by Archaeoglobus fulgidus neelaredoxin.

Neelaredoxin is a mononuclear iron protein widespread among prokaryotic anaerobes and facultative aerobes, including human pathogens. It has superoxide scavenging activity, but the exact mechanism by which this process occurs has been controversial. In this report, we present the study of the reaction of superoxide with the reduced form of neelaredoxin from the hyperthermophilic archaeon Archaeoglobus fulgidus by pulse radiolysis. This protein reduces superoxide very efficiently (k = 1.5 x 10(9) m(-1)s(-1)), and the dismutation activity is rate-limited, in steady-state conditions, by the much slower superoxide oxidation step. These data show unambiguously that the superfamily of neelaredoxin-like proteins (including desulfoferrodoxin) presents a novel type of reactivity toward superoxide, a result of particular relevance for the understanding of both oxygen stress response mechanisms and, in particular, how pathogens may respond to the oxidative burst produced by the defense cells in eukaryotes. The actual in vivo functioning of these enzymes will depend strongly on the cell redox status. Further insight on the catalytic mechanism was obtained by the detection of a transient intermediate ferric species upon oxidation of neelaredoxin by superoxide, detectable by visible spectroscopy with an absorption maximum at 610 nm, blue-shifted approximately 50 nm from the absorption of the resting ferric state. The role of the iron sixth ligand, glutamate-12, in the reactivity of neelaredoxin toward superoxide was assessed by studying two site-directed mutants: E12Q and E12V.

The genetic organization of Desulfovibrio desulphuricans ATCC 27774 bacterioferritin and rubredoxin-2 genes: involvement of rubredoxin in iron metabolism.

The anaerobic bacterium Desulfovibrio desulphuricans ATCC 27774 contains a unique bacterioferritin, isolated with a stable di-iron centre and having iron-coproporphyrin III as its haem cofactor, as well as a type 2 rubredoxin with an unusual spacing of four amino acid residues between the first two binding cysteines. The genes encoding for these two proteins were cloned and sequenced. The deduced amino acid sequence of the bacterioferritin shows that it is among the most divergent members of this protein family. Most interestingly, the bacterioferritin and rubredoxin-2 genes form a dicistronic operon, which reflects the direct interaction between the two proteins. Indeed, bacterioferritin and rubredoxin-2 form a complex in vitro, as shown by the significant increase in the anisotropy and decay times of the fluorescence of rubredoxin-2 tryptophan(s) when mixed with bacterioferritin. In addition, rubredoxin-2 donates electrons to bacterioferritin. This is the first identification of an electron donor to a bacterioferritin and shows the involvement of rubredoxin-2 in iron metabolism. Furthermore, analysis of the genomic data for anaerobes suggests that rubredoxins play a general role in iron metabolism and oxygen detoxification in these prokaryotes.

In the facultative sulphate/nitrate reducer Desulfovibrio desulfuricans ATCC 27774, the nine-haem cytochrome c is part of a membrane-bound redox complex mainly expressed in sulphate-grown cells.

The bacterium Desulfovibrio desulfuricans ATCC 27774 belongs to the group of sulphate reducers also capable of utilising nitrate as its terminal electron acceptor for anaerobic growth. One of the complex multihaem proteins found in nitrate- or sulphate-grown cells of Desulfovibrio desulfuricans ATCC 27774 is the nine-haem cytochrome c. The present work shows that the gene encoding for Desulfovibrio desulfuricans ATCC 27774 nine-haem cytochrome c is part of an operon formed by the gene cluster 9hcA-D. Besides 9hcA, the gene encoding for the nine-haem cytochrome c, genes 9hcB to D encode for a protein containing four [4Fe-4S](2+/1+) centres, for a dihaem transmembrane cytochrome b and for an unknown hydrophobic protein, respectively. The four proteins have a predicted topology that is in accordance with the formation of a membrane-bound redox complex. Furthermore, the transcriptional studies show that not only the expression of the 9HcA-D complex is dependent on the growth phase, but also is markedly increased in sulphate-grown cells.

NADH oxidase activity of mitochondrial apoptosis-inducing factor.

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein, which translocates to the nucleus during apoptosis and causes chromatin condensation and large scale DNA fragmentation. Here we report the biochemical characterization of AIF's redox activity. Natural AIF purified from mitochondria and recombinant AIF purified from bacteria (AIFDelta1-120) exhibit NADH oxidase activity, whereas superoxide anion (O(2)(-)) is formed. AIFDelta1-120 is a monomer of 57 kDa containing 1 mol of noncovalently bound FAD/mol of protein. ApoAIFDelta1-120, which lacks FAD, has no NADH oxidase activity. However, native AIFDelta1-120, apoAIFDelta1-120, and the reconstituted (FAD-containing) holoAIFDelta1-120 protein exhibit a similar apoptosis-inducing potential when microinjected into the cytoplasm of intact cells. Inhibition of the redox function, by external addition of superoxide dismutase or covalent derivatization of FAD with diphenyleneiodonium, failed to affect the apoptogenic function of AIFDelta1-120 assessed on purified nuclei in a cell-free system. Conversely, blockade of the apoptogenic function of AIFDelta1-120 with the thiol reagent para- chloromercuriphenylsulfonic acid did not affect its NADH oxidase activity. Altogether, these data indicate that AIF has a marked oxidoreductase activity which can be dissociated from its apoptosis-inducing function.

[NiFe] hydrogenase from Desulfovibrio desulfuricans ATCC 27774: gene sequencing, three-dimensional structure determination and refinement at 1.8 A and modelling studies of its interaction with the tetrahaem cytochrome c3.

The primary and three-dimensional structures of a [NiFe] hydrogenase isolated from D. desulfitricans ATCC 27774 were determined, by nucleotide analysis and single-crystal X-ray crystallography. The three-dimensional structural model was refined to R=0.167 and Rfree=0.223 using data to 1.8 A resolution. Two unique structural features are observed: the [4Fe-4S] cluster nearest the [NiFe] centre has been modified [4Fe-3S-3O] by loss of one sulfur atom and inclusion of three oxygen atoms; a three-fold disorder was observed for Cys536 which binds to the nickel atom in the [NiFe] centre. Also, the bridging sulfur atom that caps the active site was found to have partial occupancy, thus corresponding to a partly activated enzyme. These structural features may have biological relevance. In particular, the two less-populated rotamers of Cys536 may be involved in the activation process of the enzyme, as well as in the catalytic cycle. Molecular modelling studies were carried out on the interaction between this [NiFe] hydrogenase and its physiological partner, the tetrahaem cytochrome c3 from the same organism. The lowest energy docking solutions were found to correspond to an interaction between the haem IV region in tetrahaem cytochrome c3 with the distal [4Fe-4S] cluster in [NiFe] hydrogenase. This interaction should correspond to efficient electron transfer and be physiologically relevant, given the proximity of the two redox centres and the fact that electron transfer decay coupling calculations show high coupling values and a short electron transfer pathway. On the other hand, other docking solutions have been found that, despite showing low electron transfer efficiency, may give clues on possible proton transfer mechanisms between the two molecules.

A membrane-bound cytochrome c3: a type II cytochrome c3 from Desulfovibrio vulgaris Hildenborough.

A new tetraheme cytochrome c3 was isolated from the membranes of Desulfovibrio vulgaris Hildenborough (DvH). This cytochrome has a molecular mass of 13.4 kDa and a pI of 5.5 and contains four heme c groups with apparent reduction potentials of -170 mV, -235 mV, -260 mV and -325 mV at pH 7.6. The complete sequence of the new cytochrome, retrieved from the preliminary data of the DvH genome, shows that this cytochrome is homologous to the "acidic" cytochrome c3 from Desulfovibrio africanus (Da). A model for the structure of the DvH cytochrome was built based on the structure of the Da cytochrome. Both cytochromes share structural features that distinguish them from other cytochrome c3 proteins, such as a solvent-exposed heme 1 surrounded by an acidic surface area, and a heme 4 which lacks most of the surface lysine patch proposed to be the site of hydrogenase interaction in other cytochrome c3 proteins. Furthermore, in contrast to previously discovered cytochrome c3 proteins, the genes coding for these two cytochromes are adjacent to genes coding for two membrane-associated FeS proteins, which indicates that they may be part of membrane-bound oxidoreductase complexes. Altogether these observations suggest that the DvH and Da cytochromes are a new type of cytochrome c3 proteins (Type II: TpII-c3) with different redox partners and physiological function than the other cytochrome c3 proteins (Type I: TpI-c3). The DvH TpII-c3 is reduced at considerable rates by the two membrane-bound [NiFe] and [NiFeSe] hydrogenases, but catalytic amounts of TpI-c3 increase these rates two- and fourfold, respectively. With the periplasmic [Fe] hydrogenase TpII-c3 is reduced much slower than TpI-c3, and no catalytic effect of TpI-c3 is observed.

Oxygen detoxification in the strict anaerobic archaeon Archaeoglobus fulgidus: superoxide scavenging by neelaredoxin.

Archaeoglobus fulgidus is a hyperthermophilic sulphate-reducing archaeon. It has an optimum growth temperature of 83 degrees C and is described as a strict anaerobe. Its genome lacks any homologue of canonical superoxide (O2.-) dismutases. In this work, we show that neelaredoxin (Nlr) is the main O2.- scavenger in A. fulgidus, by studying both the wild-type and recombinant proteins. Nlr is a 125-amino-acid blue-coloured protein containing a single iron atom/molecule, which in the oxidized state is high spin ferric. This iron centre has a reduction potential of +230 mV at pH 7.0. Nitroblue tetrazolium-stained gel assays of cell-soluble extracts show that Nlr is the main protein from A. fulgidus which is reactive towards O2.-. Furthermore, it is shown that Nlr is able to both reduce and dismutate O2.-, thus having a bifunctional reactivity towards O2.-. Kinetic and spectroscopic studies indicate that Nlr's superoxide reductase activity may allow the cell to eliminate O2.- quickly in a NAD(P)H-dependent pathway. On the other hand, Nlr's superoxide dismutation activity will allow the cell to detoxify O2.- independently of the cell redox status. Its superoxide dismutase activity was estimated to be 59 U mg-1 by the xanthine/xanthine oxidase assay at 25 degrees C. Pulse radiolysis studies with the isolated and reduced Nlr proved unambiguously that it has superoxide dismutase activity; at pH 7.1 and 83 degrees C, the rate constant is 5 x 106 M-1 s-1. Besides the superoxide dismutase activity, soluble cell extracts of A. fulgidus also exhibit catalase and NAD(P)H/oxygen oxidoreductase activities. By putting these findings together with the entire genomic data available, a possible oxygen detoxification mechanism in A. fulgidus is discussed.

Cloning, sequencing and expression of the tetraheme cytochrome c(3) from Desulfovibrio gigas.

The gene encoding the tetraheme cytochrome c(3) from Desulfovibrio gigas was cloned and sequenced from a 2.7-kb EcoRI-PstI insert of D. gigas DNA. The derived amino acid sequence showed that the D. gigas cytochrome c(3) is synthesized as a precursor protein with an N-terminal signal peptide sequence of 25 residues and allowed the correction of the previous reported amino acid sequence (Matias et al. Protein Science 5 (1996) 1342-1354). Expression in D. vulgaris (Hildenborough) was possible by conjugal transfer of a recombinant broad-host-range vector pSUP104 containing a SmaI fragment of the D. gigas cytochrome c(3) gene. Biochemical, immunological and spectroscopic analysis of the purified protein showed that the recombinant cytochrome is identical to that isolated from D. gigas.

Expression of a Desulfovibrio tetraheme cytochrome c in Escherichia coli.

A tetraheme cytochrome c was successfully overexpressed for the first time in Escherichia coli. Desulfovibrio desulfuricans ATCC 27774 tetraheme cytochrome c(3) was expressed in aerobically grown Escherichia coli cotransformed with Escherichia coli ccm gene cluster (Arslan et al. (1998) Bioch. Biophys. Res. Commun. 251, 744-747). The analysis of the produced cytochrome showed that the signal peptide was correctly cleaved, the four heme groups were inserted and the electronic structure around the heme irons was conserved, i.e., the recombinant tetraheme cytochrome was identical to that isolated from the native source. Contradicting previous results which indicated that Escherichia coli was only capable of producing apocytochrome c(3) (Pollock et al. (1989) J. Gen. Microbiol. 135, 2319-2328), the present work proves unequivocally that the holoform can also be obtained.

Nine-haem cytochrome c from Desulfovibrio desulfuricans ATCC 27774:primary sequence determination, crystallographic refinement at 1.8 and modelling studies of its interaction with the tetrahaem cytochrome c3.

A monomeric nine-haem cytochrome c (9Hcc) with 292 amino acid residues was isolated from cells of the sulfate- and nitrate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774 grown under both nitrate- and sulfate-respiring conditions. The nucleotide sequence encoding the 292 residues was determined, allowing the correction of about 10% of the previous primary structure, determined from 1.8 A electron density maps. The refinement at 1.8 A resolution of the structural model was completed, giving an R-value of 16.5%. The nine haem groups are arranged into two tetrahaem clusters, located at both ends of the molecule, with Fe-Fe distances and local protein fold very similar to tetrahaem cytochromes c3, and the extra haem is located asymmetrically between the two regions. The new primary sequence determination confirmed the 39% sequence homology found between this cytochrome and the C-terminal region (residues 229-514) of the high-molecular-weight cytochrome c (Hmc) from D. vulgaris Hildenborough, providing strong evidence of structural similarity between 9Hcc and the C-terminal region of Hmc. The interaction between 9Hcc and the tetrahaem cytochrome c3 from the same organism was studied by modelling methods, and the results suggest that a specific interaction is possible between haem 4 of tetrahaem cytochrome c3 and haem 1 or haem 2 of 9Hcc, in agreement with previous kinetic experiments which showed the catalytic effect of the tetrahaem cytochrome c3 upon the reduction of 9Hcc by the [NiFe] hydrogenase from D. desulfuricans ATCC 27774. These studies suggest a role for 9Hcc as part of the assembly of redox proteins involved in recycling the molecular hydrogen released by the cell as a result of substrate oxidation.

Crystal structure determination at 1.4 A resolution of ferredoxin from the green alga Chlorella fusca.

BACKGROUND: [2Fe-2S] ferredoxins, also called plant-type ferredoxins, are low-potential redox proteins that are widely distributed in biological systems. In photosynthesis, the plant-type ferredoxins function as the central molecule for distributing electrons from the photolysis of water to a number of ferredox-independent enzymes, as well as to cyclic photophosphorylation electron transfer. This paper reports only the second structure of a [2Fe-2S] ferredoxin from a eukaryotic organism in its native form. RESULTS: Ferredoxin from the green algae Chlorella fusca has been purified, characterised, crystallised and its structure determined to 1.4 A resolution - the highest resolution structure published to date for a plant-type ferredoxin. The structure has the general features of the plant-type ferredoxins already described, with conformational differences corresponding to regions of higher mobility. Immunological data indicate that a serine residue within the protein is partially phosphorylated. A slightly electropositive shift in the measured redox potential value, -325 mV, is observed in comparison with other ferredoxins. CONCLUSIONS: This high-resolution structure provides a detailed picture of the hydrogen-bonding pattern around the [2Fe-2S] cluster of a plant-type ferredoxin; for the first time, it was possible to obtain reliable error estimates for the geometrical parameters. The presence of phosphoserine in the protein indicates a possible mechanism for the regulation of the distribution of reducing power from the photosynthetic electron-transfer chain.

Sequencing the gene encoding desulfovibrio desulfuricans ATCC 27774 nine-heme cytochrome c.

Contradicting early suggestions, the sequencing of the gene encoding the Desulfovibrio desulfuricans (ATCC 27774) nine-heme cytochrome c proves that this cytochrome is not the product of the degradation of the 16-heme containing cytochrome c [Coelho et al. (1996) Acta Cryst. D52, 1202-1208]. However, preliminary data indicate that the cytochrome gene is part of an operon similar to the DvH hmc operon, which contains the gene coding for the 16-heme cytochrome c [Rossi et al. (1993) J. Bacteriol. 175, 4699-4711]. Also, the amino acid sequence deduced from the DNA sequence shows four residues in the C-terminal not predicted in the amino acid sequence obtained by X-ray methods [Matias et al. (1999) Structure 7, 119-130].

Replacement of lysine 45 by uncharged residues modulates the redox-Bohr effect in tetraheme cytochrome c3 of Desulfovibrio vulgaris (Hildenborough).

The structural basis for the pH dependence of the redox potential in the tetrahemic Desulfovibrio vulgaris (Hildenborough) cytochrome c3 was investigated by site-directed mutagenesis of charged residues in the vicinity of heme I. Mutation of lysine 45, located in the neighborhood of the propionates of heme I, by uncharged residues, namely threonine, glutamine and leucine, was performed. The replacement of a conserved charged residue, aspartate 7, present in the N-terminal region and near heme I was also attempted. The analysis of the redox interactions as well as the redox-Bohr behavior of the mutated cytochromes c3 allowed the conclusion that residue 45 has a functional role in the control of the pKa of the propionate groups of heme I and confirms the involvement of this residue in the redox-Bohr effect.

Purification and preliminary characterization of three c-type cytochromes from Pseudomonas nautica strain 617.

Three c-type cytochromes, namely cytochrome c553, cytochrome c553(548) and cytochrome c', were purified from the marine denitrifying bacterium Pseudomonas nautica strain 617. These three monohemic cytochromes present in small amounts were preliminarily characterized by physiochemical and spectroscopic techniques. The visible and the 1H-NMR spectra show that cytochrome c553 and cytochrome c553(548) have histidine-methionine as iron axial ligands. Cytochrome c553 and cytochrome c553(548) have mid-point redox potentials of +269 mV and +223 mV, at pH 7.6, and their molecular masses are 14 kDa and 17 kDa, respectively. Cytochrome c' has a molecular mass of 21 kDa and its visible spectrum is typical of a high spin heme.

Replacement of methionine as the axial ligand of Achromobacter cycloclastes cytochrome c554 at high pH values revealed by absorption, EPR and MCD spectroscopy.

Cytochrome c554 from the denitrifying bacterium Achromobacter cycloclastes is a monoheme class II c-type cytochrome with a His-Met axial coordination at neutral pH. The amino acid composition and the N-terminal sequence of the cytochrome have been determined. Subsequent determination of the pH-dependence of the redox potential and examination of the EPR and MCD spectra of ferricytochrome c554 revealed a new form at high pH values made apparent with both spectroscopies. These observations are consistent with the presence of lysine as the axial ligand for which methionine substitutes at high pH values.

Physico-chemical and spectroscopic properties of the monohemic cytochrome C552 from Pseudomonas nautica 617.

A c-type monohemic ferricytochrome C552 (11 kDa) was isolated from the soluble extract of a marine denitrifier, Pseudomonas nautica strain 617, grown under anaerobic conditions with nitrate as final electron acceptor. The NH2-terminal sequence and the amino acid composition of the cytochrome were determined. The heme iron of the cytochrome C552 has histidine-methionine as axial ligands, and a pH-dependent mid-point redox potential, equal to 250 mV at pH 7.6. The presence of methionine was demonstrated by visible, EPR and NMR spectroscopies. The assignment of most of the hemic protons was performed applying two-dimensional NOE spectroscopy (NOESY), and the aromatic region was assigned through two-dimensional correlated spectroscopy (COSY) experiments. The EPR spectrum of the oxidised form of the cytochrome C552 is typical of a low-spin ferric heme.

Characterization of the dihemic cytochrome c549 from the marine denitrifying bacterium Pseudomonas nautica 617.

A dihemic ferricytochrome c549 (21 kDa) was purified and characterized from cells of the marine denitrifier Pseudomonas nautica strain 617. Several spectroscopic techniques, including UV-visible, NMR and EPR spectroscopies were applied to the characterization of this cytochrome. The visible and the 1H-NMR spectra show that both hemes have histidine-methionine as axial ligands. The dihemic cytochrome c549 has mid-point redox potentials of +230 mV and +250 mV, at pH 7.6 and its NH2-terminal sequence presents a high degree of similarity with those of cytochromes c4. The EPR studies allowed the determination of the orientation between the two axial ligands, indicating an axial ligand field for one of the hemes of cytochrome c549 and a rhombic symmetry for the other heme.

NMR and EPR studies on a monoheme cytochrome c550 isolated from Bacillus halodenitrificans.

A c-type monoheme ferricytochrome c550 (9.6 kDa) was isolated from cells of Bacillus halodenitrificans sp.nov., grown anaerobically as a denitrifier. The visible absorption spectrum indicates the presence of a band at 695 nm characteristic of heme-methionine coordination. The midpoint redox potential was determined at several pH values by visible spectroscopy. The redox potential at pH 7.6 is 138 mV. When studied by 1H-NMR spectroscopy as a function of pH, the spectrum shows a pH dependence with pKa values of 6.0 and 11.0. According to these pKa values, three forms designated as I, II and III can be attributed to cytochrome c550. The first pKa is probably associated with protonation of the propionate groups. The second pKa value introduces a larger effect in the 1H-NMR spectrum and is probably due to the ionisation of the axial histidine. Studies of temperature variation of the 1H-NMR spectra for both the ferrous and ferri forms of the cytochrome were performed. Heme meso protons, the heme methyl groups, the thioether protons, two protons from a propionate and the methylene protons from the axial methionine were identified in the reduced form. The heme methyl resonances of the ferri form were also assigned. EPR spectroscopy was also used to probe the ferric heme environment. A signal at gmax approximately 3.5 at pH 7.5 was observed indicating an almost axial heme environment. At higher pH values the signal at gmax approximately 3.5 converts mainly to a signal at g approximately 2.96. The pKa associated with this change is around 11.3. The N-terminal sequence of this cytochrome was determined and compared with known amino acid sequences of other cytochromes.