Structure and Characterization of Ectothiorhodospira vacuolata Cytochrome b 558, a Prokaryotic Homologue of Cytochrome b 5 *

A soluble cytochrome b 558from the purple phototropic bacterium Ectothiorhodospira vacuolata was completely sequenced by a combination of automated Edman degradation and mass spectrometry. The protein, with a measured mass of 10,094.7 Da, contains 90 residues and binds a single protoheme. Unexpectedly, the sequence shows homology to eukaryotic cytochromesb 5. As no prokaryotic homologue had been reported so far, we developed a protocol for the expression, purification, and crystallization of recombinant cytochromeb 558. The structure was solved by molecular replacement to a resolution of 1.65 Å. It shows that cytochromeb 558 is indeed the first bacterial cytochromeb 5 to be characterized and differs from its eukaryotic counterparts by the presence of a disulfide bridge and a four-residue insertion in front of the sixth ligand (histidine). Eukaryotes contain a variety of b 5 homologues, including soluble and membrane-bound multifunctional proteins as well as multidomain enzymes such as sulfite oxidase, fatty-acid desaturase, nitrate reductase, and lactate dehydrogenase. A search of theMycobacterium tuberculosis genome showed that a previously unidentified gene encodes a fatty-acid desaturase with an N-terminalb 5 domain. Thus, it may provide another example of a bacterial b 5 homologue.

A soluble cytochrome b 558 from the purple phototropic bacterium Ectothiorhodospira vacuolata was completely sequenced by a combination of automated Edman degradation and mass spectrometry. The protein, with a measured mass of 10,094.7 Da, contains 90 residues and binds a single protoheme. Unexpectedly, the sequence shows homology to eukaryotic cytochromes b 5 . As no prokaryotic homologue had been reported so far, we developed a protocol for the expression, purification, and crystallization of recombinant cytochrome b 558 . The structure was solved by molecular replacement to a resolution of 1.65 Å. It shows that cytochrome b 558 is indeed the first bacterial cytochrome b 5 to be characterized and differs from its eukaryotic counterparts by the presence of a disulfide bridge and a four-residue insertion in front of the sixth ligand (histidine). Eukaryotes contain a variety of b 5 homologues, including soluble and membrane-bound multifunctional proteins as well as multidomain enzymes such as sulfite oxidase, fattyacid desaturase, nitrate reductase, and lactate dehydrogenase. A search of the Mycobacterium tuberculosis genome showed that a previously unidentified gene encodes a fatty-acid desaturase with an N-terminal b 5 domain. Thus, it may provide another example of a bacterial b 5

homologue.
Soluble cytochromes from purple phototropic bacteria are almost invariably found to be of the c-type with covalently bound heme (1, 2). One of the few previously recognized b-type soluble cytochromes in purple bacteria is a minor component and has a large molecular mass (3). It is now known to be a bacterioferritin and to be widespread in bacteria as a whole (4).
The best characterized soluble cytochrome b is the b 562 from Escherichia coli, with a molecular mass of 12 kDa and a redox potential of ϩ200 mV (5)(6)(7). The three-dimensional structure is an antiparallel four-helix bundle, similar to that of cytochrome cЈ (8).
Kusche and Trü per (9) found a small, soluble cytochrome b 558 in Ectothiorhodospira shaposhnikovii, in addition to cytochromes c 554 and cЈ. We found very similar proteins in the related species, Ectothiorhodospira vacuolata. High potential iron protein isozymes were also purified from these two species (10), and the sequences were found to be very similar (11). 1 In fact, they are so similar that the two bacteria may be considered to be strains of the same species. The cytochrome b 558 from E. shaposhnikovii was reported to have a mass of 15.8 kDa and a redox potential of Ϫ210 mV (9). The immediate goal of this study was to identify the cytochrome and to determine its primary and tertiary structures as a contribution to the determination of its functional role.

MATERIALS AND METHODS
Protein Isolation and Modification-Native cytochrome b 558 from E. vacuolata was prepared essentially as described by Kusche and Trü per (9). The amino acid sequence was determined with 1.9 mg of protein. Prior to sequencing, the cysteines of the protein were reduced with dithiothreitol and pyridylethylated as described by Friedman et al. (12). Heme and reagents were removed from the denatured protein by gel filtration on a Sephadex G-25F column (22 ϫ 1.5 cm; Amersham Pharmacia Biotech, Uppsala, Sweden) equilibrated in and eluted with 5% formic acid.
Protein Sequence Analysis-Digestion with Lys-C endoproteinase (Wako, Osaka, Japan) was performed on 18.6 nmol of pyridylethylated apoprotein in 100 mM ammonium bicarbonate buffer, pH 8.0, at an enzyme/substrate ratio of 1:30 for 2 h at 37°C. The same conditions were used for Glu-C endoproteinase (Roche Molecular Biochemicals) digestion, but the digestion mixture was incubated for 4 h at 37°C.
Peptides obtained after enzymatic digestions were separated by reversed-phase high performance liquid chromatography using a Pep-S C2/C18 column (250 ϫ 4 mm; Amersham Pharmacia Biotech). The chromatographic equipment consisted of two Model 6000A chromatographic pumps (Waters, Milford, MA), an injector with a loop of 100-l volume (Rheodyne, Inc., Cotati, CA), and a UV detector set at 220 nm (Pye Unicam, Cambridge, MA). The solvents used were 0.1% trifluoroacetic acid in water (solvent A) and 0.1% trifluoroacetic acid in acetonitrile (solvent B) at a flow rate of 1 ml/min. Automated N-terminal sequence and electrospray ionization mass analyses were performed as described (13).
PCR 2 Strategy-The gene for the E. vacuolata cytochrome b 558 was obtained via PCR amplification (Vent polymerase, New England Biolabs Inc., Beverly, MA) using degenerate primers based on the amino acid sequence of the protein. The sequence for the amino-terminal primer (based on the first six residues of the protein sequence) was 5Ј-AAYGARACNGARGCNACN-3Ј, and that of the carboxyl-terminal primer (based on sequence 84 -90) was 5Ј-HTANCARAGRAGITC-NCANGG-3Ј. The PCR fragment was cloned in the vector pGEM-T (Promega, Madison, WI), and the resulting construct (pGEMT-b 558 ) was verified by sequence analysis.
Construction of the Overexpression Plasmid-To achieve secretion of cytochrome b 558 into the periplasmic space of E. coli, the gene was fused with the nucleotide sequence encoding the signal peptide (sOmpA) of the bacterial outer membrane protein OmpA. Thus, the b 558 gene was inserted after the sOmpA leader sequence using the NaeI and HindIII restriction sites of plasmid pT10sOmpArPDI (14 -16). As verified by DNA sequence analysis, sOmpA was fused in-frame to the mature b 558 . The hybrid gene encoding the fusion sOmpA/b 558 was transferred from pT10sOmpAb 558 to pLPPsOmpArPDI using the restriction sites XbaI and HindIII and also to pQE60 (QIAGEN Inc.) as a NcoI/HindIII fragment. The b 558 gene was also cloned without signal sequence in the pQE60 plasmid. The resulting constructs (pLPPsOmpAb 558 , pQE60s-OmpAb 558 , and pQE60b 558 ) were verified by nucleotide sequencing.
Production of Recombinant Cytochrome b 558 in E. coli-The bacterial strain MC1061 (17) harboring the expression plasmid pLPPsOmpAb 558 was grown in LB medium supplemented with carbenicillin (100 g/ml). After 16 h of incubation, cells were harvested by centrifugation (4000 rpm, 10 min, 4°C), and the periplasmic fraction was collected after treatment as described by Koshland and Botstein (18). After SDSpolyacrylamide gel electrophoresis and electroblotting, the N-terminal amino acid sequence was determined to check for the correct removal of the signal peptide from the secreted protein.
Protein Purification-Purification of recombinant cytochrome b 558 was achieved by a two-step procedure utilizing ion-exchange and sizeexclusion chromatographies. The periplasmic protein fraction was first loaded on a Q-Sepharose Fast Flow column (1-ml bed volume; Amersham Pharmacia Biotech) in 50 mM Tris-Cl, pH 7.4. The cytochrome b 558 fraction was completely retained on the column and was eluted with 0.35 M NaCl and 50 mM Tris-Cl, pH 7.4. The pooled protein was further purified on an HL-Superdex 75 column (1.5 ϫ 60 cm) connected to an Ä kta chromatographic system (Amersham Pharmacia Biotech). The cytochrome b 558 -containing fractions were pooled and concentrated using Vivaspin filters (molecular mass cutoff of 5 kDa). Mass spectroscopy and N-terminal amino acid sequence analysis were used to judge the purity of the preparation.
Characterization of Recombinant Cytochrome b 558 from E. coli-Protein samples were subjected to reducing SDS-polyacrylamide gel electrophoresis (19) and stained with Coomassie Blue or silver (20). Native electrophoresis was used to detect the presence of the heme group by peroxidase reaction (21). Total protein concentration was determined by the method of Bradford (22) using the Bio-Rad 500-0006 kit with bovine serum albumin as a standard curve.
Crystallization and X-ray Data Collection-The protein solution was concentrated to 10 mg/ml and dialyzed against 10 mM Tris-Cl, pH 7.4, to set up crystallization trials using the hanging drop diffusion method. The protein crystallized in 4 M NaCl, pH 5.6, and 0.1 M MES. Crystals grew over a period of several days to a maximum size of 0.2 ϫ 0.05 ϫ 0.05 mm. The crystals belong to space group P3 2 21 with unit cell parameters a and b ϭ 46.406 Å and c ϭ 91.66 Å. Assuming a molecular mass of 10,094.7 Da, as determined by electrospray ionization mass spectrometry, and with one molecule in the asymmetric unit, the V m value is 3.42, corresponding to a solvent content of 63.8%. These values are within the observed range for protein crystals (23). Data collection was performed at the LURE Synchrotron (Orsay, France) on beam-line DW32 ( ϭ 0.97 Å) using a Mar-Research Mar345 imaging plate detector. The x-ray diffraction data were indexed, processed, scaled, and merged using DENZO and SCALEPACK (24). A total of 92,730 measurements from 14,370 unique reflections were recorded, and R merge was 5.9% for the data between 15 and 1.65 Å with a completeness of 99.6%.

RESULTS AND DISCUSSION
Protein Sequence Determination-The complete amino acid sequence of cytochrome b 558 (Fig. 1, sequence 1) was obtained by N-terminal sequence analysis of the native protein up to position 16 and of overlapping peptides obtained from Glu-C and Lys-C enzymatic digestions on pyridylethylated apoprotein. Peptides obtained from these two digestions covered the complete sequence. Fragment S12B contained an intramolecular disulfide bridge, which was found to connect Cys 24 and Cys 53 of the complete protein sequence. Mass data on the different peptides confirming the sequence data are summa-rized in Table I. The measured mass of native E. vacuolata cytochrome b 558 is 10,094.7 Da, which is in agreement with the calculated mass of 10,095.3 Da (Fig. 2).
The three-dimensional structures clearly show that the tetrapeptides do not align.
desaturases of plants and animals. We developed the strategy to determine the crystal structure of cytochrome b 558 to identify it more conclusively. Because we had insufficient native protein for crystallization trials, we prepared recombinant protein for these studies.
Expression of Recombinant Cytochrome b 558 in E. coli-Information obtained from back-translation of the amino acid sequence was used to design degenerate oligonucleotide primers for PCR against the E. vacuolata genomic DNA template. A PCR fragment of the appropriate size and sequence was obtained, showing that it was the correct product derived from the b 558 gene. The periplasmic protein fractions from the pQE60sOmpAb 558 , pT10sOmpAb 558 , and pLPPsOmpAb 558 constructs were prepared from E. coli cells, grown aerobically at 37°C until saturation (A 600 ϭ 2.3). Recombinant cytochrome b 558 was expressed from plasmid pLPPsOmpAb 558 only when isopropyl-␤-D-thiogalactopyranoside was omitted. When isopropyl-␤-D-thiogalactopyranoside was added, expression was considerably suppressed. The other plasmids did not produce any detectable amounts of cytochrome. Therefore, pLPPsOmpAb 558 was used for expression of cytochrome b 558. SDS-polyacrylamide gel electrophoresis analysis (data not shown) showed cytochrome b 558 to be the major component in the periplasmic protein fraction. N-terminal amino acid sequence analysis revealed the expected sequence of mature cytochrome b 558 , confirming the correct processing of the signal peptide by an E. coli signal peptidase.
To ascertain that recombinant cytochrome b 558 did incorporate a heme cofactor, the tetramethylbenzidine-hydrogen peroxide test was performed. On native polyacrylamide gels, a unique band was indeed visualized due to the peroxidase activity of the cytochrome. The dithionite-reduced and air-oxi- dized difference absorption spectra indicated that the oxidized Soret peak maximum was at 414 nm; and upon reduction, the ␣-peak was at 557.5 nm, and the Soret peak was shifted to 424.5 nm and increased in magnitude (Fig. 3), confirming that it had native redox properties.
Purification of Cytochrome b 558 from E. coli Periplasm-Cytochrome b 558 was successfully purified from the osmotic shock fluid by ion-exchange chromatography and gel filtration. Analysis using isoelectric focusing gels showed that cytochrome b 558 was retained at a pH of 4.5, which is in accordance with the calculated pI value of 4.75 based upon the sequence (data not shown). On a Q-Sepharose Fast Flow column, cytochrome b 558 bound very efficiently at pH 7.4. The eluted protein was judged to be 90% pure. To remove minor contaminating proteins, the cytochrome b 558 fraction was loaded on an HL-Superdex 75 column in 100 mM Tris-Cl, pH 7.4, and 100 mM NaCl. Analysis on silver-stained SDS-polyacrylamide gels and mass spectrometry showed the protein to be fully pure.
Three-dimensional Structure Analysis-The crystal structure of cytochrome b 558 was solved by molecular replacement with bovine cytochrome b 5 (Protein Data Bank code 1CYO) as the search model. Molecular replacement calculations were performed using AmoRe (25). Refinement was carried out using the maximum likelihood-based program REFMAC (26). Ordered water molecules were included by selecting the peaks based on F o Ϫ F c difference Fourier maps contoured at 3.0 and the 2F o Ϫ F c density contoured at 1 (Fig. 4). Residues and side chains not visible in the electron density were omitted from the model. For data collection and refinement statistics, see Table II.
The polypeptide chain of cytochrome b 558 is folded in five short ␣-helices and five ␤-strands involved in a twisted sheet structure (Fig. 5). This is the same overall three-dimensional structure as for eukaryotic cytochrome b 5 (31). The first five N-terminal and the last four C-terminal residues are not visible in the electron density. Most ␣-helices are very short, having incomplete ␣-helical geometry (31). In Fig. 1, we show the where I is the observed intensity, and ϽIϾ is the average intensity of multiple observations of symmetry-related reflections. (27) is the same as R cryst , but was calculated using a separate validation set of reflections that was excluded from the refinement process. c As calculated by the program PROCHECK (28). d Root mean square deviation. structure-based alignment of cytochrome b 558 with cytochrome b 5 sequences for which the three-dimensional structures are already known. All of them are soluble or membrane-bound redox proteins, although in some, the cytochrome b is a domain within a larger enzyme structure (such as, for example, sulfite oxidase and flavocytochrome-b 2 :lactate dehydrogenase).
Structure of Cytochrome b 558 and Comparison with Homologous Proteins-When comparing cytochrome b 558 with the cytochrome b 5 domain of Saccharomyces cerevisiae flavocytochrome b 2 (32) and with bovine microsomal cytochrome b 5 (33), it is apparent that cytochrome b 558 resembles the latter more closely, having the same orientation for both ␣-helix 5 and the heme group. The C-␣ traces of b 558 and bovine microsomal b 5 are very similar (Fig. 6A), the only major differences being situated in the loops connecting the ␣-helices involved in heme binding. A close comparison of the two molecules clearly indicates a different orientation of the heme-binding region relative to the twisted sheet structure in b 558 . This rather small movement is mediated by changes in backbone torsion angles around Gln 55 , Leu 35 , and Tyr 81 .
A structural characteristic novel to the b 5 fold in cytochrome b 558 is the disulfide bridge between two strands formed by Cys 24 and Cys 53 . Although a single cysteine is observed at position 24 (b 558 numbering) in plant cytochromes b 5 (34) and yeast flavocytochrome b 2 , most animal cytochromes b 5 lack cysteine altogether. The disulfide bridge appears to span a hydrophobic cleft on the surface of the molecule. This cleft is situated in between two hydrophobic sites of the protein as described by Mathews and co-workers (31). It functions as a sort of a channel making the heme more accessible to the solvent. Recent molecular dynamics simulations (35) as well as NMR and fluorescence studies (36) provide evidence of localized motion at the cleft area. The same authors (36, 37) studied a "de novo" incorporated disulfide bond in the b 5 protein, near the same position as in native cytochrome b 558 . Their result suggests that the engineered disulfide bond inhibits the dynamics of the cleft formation. In the case of cytochrome b 558 , the influence of the disulfide bridge on the native dynamics remains to be investigated, as does its influence on the interaction with redox partners. A comparison of the structures of cytochrome b 558 , bovine cytochrome b 5 , yeast flavocytochrome b 2 (Fig. 6B), and chicken sulfite oxidase (38, 39) (Fig. 6C), a molybdopterin cytochrome . This is illustrated in the sequence alignment of Fig. 1. It is curious that several of the insertions and deletions occur at the molecular surface around the heme where electron transfer is thought to take place. They may thus play a role in molecular recognition of reaction partners.
The heme group in cytochrome b 558 is buried in a hydrophobic cleft flanked by four short ␣-helices (␣-helices 2-5) at the sides and by the twisted ␤-sheet at the bottom (Fig. 5). In comparison with bovine b 5 , the heme group is in the so-called B-conformation, rotated 180°around the a,y-meso-carbon atoms relative to the observed A-conformation in bovine b 5 (Fig.  6A). In b 558 , the A-orientation would be less stable due to a close contact with Leu 78 , a bulky residue that replaces the smaller Ser 78 of bovine b 5 . His 42 and His 70 , extending from the side of the cavity, ligate the iron atom. Whereas in bovine cytochrome b 5 , one propionic acid group is curved toward the interior of the molecule and the other projects outward in solution, cytochrome b 558 has both heme propionates bent back toward the interior of the molecule. Both propionates form salt bridges with Lys 64 (Fig. 7), a structural property not normally encountered in cytochromes b 5 , but frequently observed in cytochromes c. Whereas propionate B is hydrogen-bonded to Tyr 66 , propionate A is hydrogen-bonded to the hydroxyl group and the peptide nitrogen of Ser 71 , the latter contact being preserved in bovine b 5 . Both Lys 64 and Tyr 66 are close to or part of the four-residue insertion in the b 558 sequence when compared with the b 5 family.
The loop connecting ␣-helices 2 and 3 at the other side of the heme plane is formed by the strictly conserved Pro 43 -Gly 44 -Gly 45 sequence in the cytochrome b 5 family. This sequence is positioned at a strategic point where chain reversal occurs, leaving no space to accommodate a side chain at positions 44 and 45. In cytochrome b 558 , however, the second glycine is replaced by another proline residue, reorienting the local back-bone conformation relative to bovine b 5 . This relocation results in a change in the hydrogen bond partner for the second imidazole nitrogen of His 42 , which is normally the carbonyl oxygen of Gly 45 , to a water molecule. The other heme ligand (His 70 ) is hydrogen-bonded to the carbonyl oxygen of Trp 61 , similar to the interaction with Phe 61 in bovine b 5 . The Phe 61 aromatic ring in cytochrome b 558 is stacked parallel to the imidazole plane of the second histidine, positioning it firmly in the heme-binding site. This residue is replaced by Trp 61 in cytochrome b 558 , with the indole group having the same structural role.
In addition to the hydrophobic core that constitutes the heme-binding site, a second and smaller hydrophobic core is situated at the other side of the twisted ␤-sheet. This core is even more compact in cytochrome b 558 , where the replacement of the bulky side chains of Ile 15 and Ile 27 by valine and alanine allows ␣-helix 1 to pack more closely to the ␤-sheet. Trp 25 is part of a conserved structural motif in the small hydrophobic core: its indole side chain is sandwiched between the imidazole ring of His 18 and a methyl group of Ile 83 . In bovine b 5 , Ser 21 is hydrogen-bonded to Arg 50 , which is in electrostatic interaction with Glu 46 . This hydrophilic interaction has a hydrophobic counterpart in b 558 , where Pro 21 is in van der Waals interaction with Leu 50 , resulting in a reorientation of the local backbone conformation around Pro 21 . In addition to the Arg 50 /Glu 46 contact, bovine b 5 has two more salt bridges between exposed side chains. These contacts do not exist in cytochrome b 558 , which has no salt bridges except for the interaction of Lys 64 with the heme propionates (see above).
Two sets of polar interactions that contribute to anchoring the wall of the heme-binding site to the bottom are conserved in both proteins. On one side of the heme, the hydroxyl group of Thr 36 is hydrogen-bonded to both the peptide amide of Cys 24 and the carboxylate of Asp 34 , whereas on the other side, Thr 58 interacts with Glu 56 and the backbone nitrogen of His 29 .
Bovine b 5 has several negatively charged residues surround- FIG. 7. Stereo representation of the heme pocket. The heme group and selected residues involved in heme binding are represented by atom colored sticks, whereas the backbone is represented by ribbons. Color coding is the same as described in the legend to Fig. 5. This figure was generated using Molscript (29) and Raster3D (30). The surface is colored according to the electrostatic potential (red, negative; blue, positive) and made transparent; the heme group is blue. This figure was generated by GRASP (41). ing the exposed heme edge. These residues are implicated in electrostatic interactions with their physiological partners. Redox partners carry a complementary positively charged ring as described in the model of Salemme and co-workers (40) for the cytochrome b 5 -cytochrome c complex. On the other hand, intramolecular interactions between the cytochrome b 2 domain and the FAD domain in flavocytochrome b 2 involve but one salt bridge interaction, all others being water-mediated hydrogen bonds or van der Waals interactions. Likewise, cytochrome b 558 has no predominant negative charge positioned around the exposed heme edge (Fig. 8). Besides Glu 68 , only the two propionic acid groups carry a negative charge in this region, and they are partially neutralized by Lys 64 . The interactions of cytochrome b 558 with hypothetical reaction partners could thus be quite different from those of cytochrome b 5 .
Possible Functional Role for Cytochrome b 558 -A biological function for E. vacuolata cytochrome b 558 is not known so far. However, we do want to propose two possibilities, which we deduced from considering the distribution of cytochrome b 5 homologues, including an analysis of the Ͼ20 bacterial and two eukaryotic genomes that are presently known.
Cytochrome b 5 had not been reported in any bacterial species until now, and b 5 genes have not been identified in any of the bacterial genomes. However, a BLAST search of the Mycobacterium tuberculosis genome (42) revealed the presence of an unidentified gene (Rv1371) with similarity to the fatty-acid desaturases of plants, animals, and fungi. That it is indeed a b 5 homologue is shown in the alignment of Fig. 1. It has the two His heme ligands located at the appropriate positions in the sequence, the Pro-Gly-Gly tripeptide after the fifth heme ligand, and Trp 22 as well as other conserved residues. It has the flavocytochrome b 2 deletion ahead of the sixth heme ligand and a two-residue insertion after helix 5. It also appears to have one of the sulfite oxidase insertions after the sixth heme ligand. The M. tuberculosis cytochrome b 5 differs from other cytochromes b 5 in having a single-residue deletion three or four residues after Trp 22 . We think there are two likely functional roles for cytochrome b 558 in E. vacuolata. It could be a subunit of a fatty-acid desaturase as found in M. tuberculosis, yeast (43), and animals such as Caenorhabditis elegans (44). An equally likely alternative because of the utilization of reduced sulfur compounds by E. vacuolata is a sulfite oxidase subunit.
Evolutionary Considerations-The present eukaryotic cytochromes b 5 can be assumed to be derived from an ancestral prokaryotic cytochrome b 5 , for which the E. vacuolata cytochrome b 558 could stand as a model. The structure of the prokaryotic cytochrome b 558 shows that this protein provides an evolutionarily stable framework that permits a considerable exchange of residues, including the amino acids at the surface as well as amino acids outlining the heme environment, causing no fundamental structural change in the polypeptide backbone. Comparing ϳ40 deposited b 5 sequences, Lederer (6) located eight invariant residues in the b 5 fold, of which two now appear not to be conserved in the prokaryotic cytochrome b 558 . The two residues that are replaced are involved in the interactions with the heme ligands (His 42 and His 70 ). The proline that replaces Gly 45 in the His-Pro-Gly-Gly loop causes slight changes in the backbone conformation and creates space for an extra water molecule in the His 42 environment. The invariant Phe 61 is replaced by tryptophan, retaining the same role in the stabilization of His 70 . Both changes do not seem to be typical of prokaryotic b 5 , as the M. tuberculosis open reading frame has Gly at position 45 and lacks an aromatic side chain at position 61 altogether.
Although it is surprising how strict the fold is conserved throughout evolution, the function of cytochrome b 5 diverged in eukaryotic organisms, and the protein has become a modular domain of several redox systems. Residues conserved throughout the b 5 family are therefore to be associated with folding of the protein and binding of a heme group and not with interacting with the redox partners. With the exception of Gly 45 , replaced by Pro 45 , all these residues are conserved in cytochrome b 558 . It is interesting to note that the E. vacuolata protein is not significantly more related to any particular functional class of eukaryotic cytochrome b 5 , whether the microsomal b 5 domain of flavocytochrome b 2 , sulfite oxidase, or fatty-acid desaturases. It is in fact almost equidistant to these four subclasses of the cytochrome b 5 family.