Identification of outer mitochondrial membrane cytochrome b5 as a modulator for androgen synthesis in Leydig cells

Outer mitochondrial membrane cytochrome b5 is an isoform of microsomal membrane cytochrome b5. In rat testes the outer mitochondrial membrane cytochrome b5 is present in both mitochondria and microsomes, whereas microsomal membrane cytochrome b5 is undetectable. Outer mitochondrial membrane cytochrome b5 present in the testis was localized in Leydig cells with cytochrome P-45017alpha, which catalyzes androgenesis therein. We therefore analyzed the functions of outer mitochondrial membrane cytochrome b5 in rat testis microsomes by using a proteoliposome system. In a low but physiological concentration of NADPH-cytochrome P-450 reductase and excess amount of progesterone, outer mitochondrial membrane cytochrome b5 stimulated the cytochrome P-45017alpha-catalyzed reactions, 17alpha-hydroxylation and C17-C20 bond cleavage. The effects were different from those by microsomal membrane cytochrome b5 as follows: preferential elevation of the 17alpha-hydroxylase activity by outer mitochondrial membrane cytochrome b5 in an amount-dependent manner versus that of the lyase activity by microsomal membrane cytochrome b5 at the low concentration, and the inhibition of both activities at the high concentration. At a low concentration of progesterone reflecting a physiological cholesterol supply, outer mitochondrial membrane cytochrome b5 elevated primarily the production of 17alpha-hydroxyprogesterone and then facilitated the conversion of the released intermediate to androstenedione. Thus, we demonstrated that outer mitochondrial membrane cytochrome b5 and not microsomal membrane cytochrome b5 functions as an activator for androgenesis in rat Leydig cells.     

Hamster hepatic cytochrome b5: purifications, immunochemical properties, and in vitro synthesis

Cytochrome b5 has been purified from hamster liver microsomes. Both Ouchterlony double-diffusion and rocket immunoelectrophoresis experiments indicate that no immuno-cross-reactivity exists between guinea-pig anti-rabbit cytochrome b5 antibody and hamster cytochrome b5. However, anti-rabbit b5 IgG inhibited both hamster microsomal NADH-cytochrome c reductase and NADPH-dependent 7-ethoxycoumarin-O-deethylase activities. Hamster cytochrome b5 stimulated several reconstituted hamster cytochrome P-450-dependent monooxygenase activities and this stimulatory effect could be inhibited by antibody against rabbit cytochrome b5. Two-dimensional iodinated tryptic peptide mapping experiments provided evidence that the polypeptide fingerprint of hamster cytochrome b5 is substantially different from the fingerprints of cytochrome b5 isolated from rabbit, rat and bovine. We also studied the in vitro synthesis of hamster cytochrome b5 from liver mRNA using a wheat germ lysate system. A 16 kDa polypeptide, which is the same size as hamster cytochrome b5, was immunoprecipitated by antibody against rabbit b5. This experiment suggested that in vitro synthesized hamster cytochrome b5 is recognized by a heterologous antibody. Thus, hamster and rabbit cytochrome b5 do share some common immuno-determinants which may be located close to the heme-binding active site.     

Cytochrome b5 reductase and cytochrome b5 support the CYP2E1-mediated activation of nitrosamines in a recombinant Ames test

With CYP2E1 in vitro both the first and the second electron of the catalytic cycle can come from cytochrome b(5) via either NADPH-cytochrome P450 reductase or NADH-cytochrome b(5) reductase, and the presence of cytochrome b(5) stimulates CYP2E1 turnover both in vitro and in vivo. To determine whether electron input via the NADH-dependent pathway was similarly functional in whole cells and necessary for the stimulation by cytochrome b(5), we constructed five plasmids designed to express human CYP2E1 in various combinations with cytochrome b(5) reductase, cytochrome b(5), and cytochrome P450 reductase. CYP2E1 activity in Salmonella typhimurium cells transformed with each plasmid was assessed by mutagenic reversion frequency in the presence of dimethylnitrosamine. A fivefold increase in reversion frequency when cytochrome b(5) was coexpressed with P450 reductase was abolished by disruption of heme-binding in cytochrome b(5) by site-directed mutagenesis (His68Ala), suggesting that electron transfer to cytochrome b(5) was necessary for the stimulation. Addition of cytochrome b(5) reductase to the cytochrome b(5)/P450 reductase coexpression plasmid did not further increase the stimulation by cytochrome b(5), but b(5) reductase could support CYP2E1 activity in the absence of P450 reductase at a level equivalent to that obtained with just CYP2E1 and P450 reductase. Neither cytochrome b(5) reductase nor cytochrome b(5) alone could support CYP2E1 activity. These results demonstrate that the cytochrome b(5) reductase/cytochrome b(5) pathway can support CYP2E1 activity in bacterial cells.     

The identification of the heat-stable microsomal protein required for methoxyflurane metabolism as cytochrome b5

Methoxyflurane is an anesthetic whose metabolism by cytochrome P-450LM2 has been shown to be dependent upon a heat-stable microsomal protein (Canova-Davis, E., and Waskell, L. A. (1982) Biochem. Biophys. Res. Commun. 108, 1264-1270). Treatment of this protein with diethylpyrocarbonate, which modifies selected amino acids, caused a dose-dependent loss in its ability to effect the metabolism of methoxyflurane by purified cytochrome P-450LM2. This protein factor has been identified as cytochrome b5 by demonstrating that cytochrome b5 and the heat-stable factor coelute during cytochrome b5 purification. Neither ferriheme nor apocytochrome b5 was able to substitute for the activating factor, while cytochrome b5 reconstituted from apocytochrome b5 and heme exhibited an activity similar to that of native b5. Examination of the cytochrome b5 molecule by computer graphics suggested that diethylpyrocarbonate did not inactivate b5 by reacting with the anionic surface of the cytochrome b5 molecule. Maximal rates of methoxyflurane metabolism were obtained at a ratio of 1:1:1 of the three proteins, cytochrome P-450LM2:reductase:cytochrome b5. In summary, it has been demonstrated that the heat-stable protein, cytochrome b5, is obligatory for the metabolism of methoxyflurane by cytochrome P-450LM2. These data also suggest that cytochrome b5 may be acting as an electron donor to P-450LM2 in the O-demethylation of methoxyflurane.     

Cytochrome b5-mediated redox cycling of estrogen

Previously, we have demonstrated microsomal cytochrome P450-catalyzed redox cycling of estrogens. In this study, we investigated the role of cytochrome b5 in redox cycling in order to obtain a full understanding of enzymatic contributions to redox reactions of estrogens. Pure cytochrome P450c and hydrogen peroxide or cumene hydroperoxide oxidized diethylstilbestrol (DES) to diethylstilbestrol-4',4"-quinone (DES Q). This oxidation by H2O2 was doubled by addition of cytochrome b5 to cytochrome P450c (molar ratio of 1:4), but did not proceed with cytochrome b5 alone. The stimulation by cytochrome b5 of the cytochrome P450c-catalyzed oxidation of DES to DES Q occurred via modulation of the Vmax of cytochrome P450c rather than of the Km. DES Q was reduced to DES by purified cytochrome b5 and NADH-dependent cytochrome b5 reductase. Pretreatment of microsomes with an antibody to cytochrome b5 reductase inhibited microsomal NADH-dependent reduction of DES Q to DES by 55%. Cytochrome b5 likely participates in the oxidation of DES to DES Q by interacting with cytochrome P450c and in the reduction of DES Q to DES by interacting with cytochrome b5 reductase. Thus, the study demonstrates that cytochrome b5 plays an active role in biological oxidation and reduction reactions.     

Chemical characterization of protein-protein interactions between cytochrome P-450 and cytochrome b5

Native cytochrome b5 interacts with either RLM5 or LM2 to form tight equimolar complexes (Kd = 250 and 540 nM, respectively) in which the content of high spin cytochrome P-450 was substantially increased. Cytochrome b5 caused 3- and 7-fold increases in the binding affinities of RLM5 and LM2 for benzphetamine, respectively, and benzphetamine decreased the apparent Kd for cytochrome b5 binding. Upon formation of the ternary complex between cytochromes P-450, b5, and benzphetamine the percentage of cytochrome P-450 in the high spin state was increased from 28 to 74 (RLM5) and from 9 to 85 (LM2). Cytochrome b5 caused 13- and 7-fold increases in the rate of RLM5- and LM2-dependent p-nitroanisole demethylation, respectively. Amino-modified (ethyl acetimidate or acetic anhydride) cytochrome b5 produced results similar to those obtained above with native cytochrome b5. In contrast, modification of as few as 5 mol of carboxyl groups/mol of amidinated cytochrome b5 resulted in both a substantial loss of the spectrally observed interactions with either cytochrome P-450 LM2 or cytochrome P-450 RLM5, and in a loss of the cytochrome b5-mediated stimulation of p-nitroanisole demethylation catalyzed by either monooxygenase. In further studies, native and fully acetylated cytochromes b5 reoxidized carbonmonoxy ferrous LM2 at least 20 times faster than amidinated, carboxyl-modified cytochrome b5 derivatives. In contrast, amidination, or acetylation of amino groups, or amidination of amino groups plus methylamidination of the carboxyl groups did not appreciably slow the rate of reduction of the cytochrome b5 by NADPH-cytochrome P-450 reductase. Collectively, the results provide strong evidence for an essential role of cytochrome b5 carboxyl groups in functional interactions with RLM5 and LM2.     

Biodiversity of the P450 catalytic cycle: yeast cytochrome b5/NADH cytochrome b5 reductase complex efficiently drives the entire sterol 14-demethylation (CYP51) reaction

The widely accepted catalytic cycle of cytochromes P450 (CYP) involves the electron transfer from NADPH cytochrome P450 reductase (CPR), with a potential for second electron donation from the microsomal cytochrome b5/NADH cytochrome b5 reductase system. The latter system only supported CYP reactions inefficiently. Using purified proteins including Candida albicans CYP51 and yeast NADPH cytochrome P450 reductase, cytochrome b5 and NADH cytochrome b5 reductase, we show here that fungal CYP51 mediated sterol 14alpha-demethylation can be wholly and efficiently supported by the cytochrome b5/NADH cytochrome b5 reductase electron transport system. This alternative catalytic cycle, where both the first and second electrons were donated via the NADH cytochrome b5 electron transport system, can account for the continued ergosterol production seen in yeast strains containing a disruption of the gene encoding CPR.     

Oxidation of cytochrome b5 reduced by ascorbic acid

The steady-state levels of aerobic and anaerobic reduction of cytochrome b5 by ascorbic acid and the initial rates of cytochrome b5 reduction in the presence of ascorbic acid and of anaerobic cytochrome P-450 reduction in the presence of NADH were used to calculate the rate constants for cytochrome b5 oxidation. The rate constant for cytochrome b5 autooxidation in the membrane is equal to that for isolated cytochrome b5, i. e., 5 X 10(-3) s-1 (37 degrees C). The rate constant for the second cytochrome b5 oxidation reaction in the membrane, i. e., electron transfer to cytochrome P-450, is equal to 140 X 10(-3) s-1 (37 degrees C).     

Kinetics of flavin semiquinone reduction of the components of the cytochrome c-cytochrome b5 complex

The kinetics of flavin semiquinone reduction of the components of the 1:1 complex formed by cytochrome c with either cytochrome b5 or a derivative of cytochrome b5 in which the heme propionates are esterified (DME-cytochrome b5) have been studied. The rate constant for the reduction of horse heart cytochrome c by the electrostatically neutral lumiflavin semiquinone (LfH) is unaffected by complexation with native cytochrome b5 at pH 7. However, complex formation with DME-cytochrome b5 (pH 7) decreases by 35% the rate constant for cytochrome c reduction by LfH. At pH 8, complex formation with native cytochrome b5 decreases the rate constant for cytochrome c reduction by LfH markedly, whereas the rate constant for cytochrome c reduction, either unbound or in the complex formed with DME-cytochrome b5, is increased 2-fold relative to pH 7. These results indicate that the accessibility of the cytochrome c heme is not the same in the complexes formed with the two cytochrome b5 derivatives and that the docking geometry of the complex formed by the two native cytochromes is pH dependent. Binding of horse heart and tuna cytochromes c to native and DME-cytochromes b5 decreases the rate constants for reduction of cytochrome c by the negatively charged flavin mononucleotide semiquinone (FMNH) by approximately 30% and approximately 40%, respectively. This finding is attributed to substantial neutralization of the positive electrostatic potential surface of cytochrome c that occurs when it binds to either form of cytochrome b5.(ABSTRACT TRUNCATED AT 250 WORDS)     

Methoxyflurane acts at the substrate binding site of cytochrome P450 LM2 to induce a dependence on cytochrome b5

Rabbit cytochrome P450 isozyme 2 requires cytochrome b5 to metabolize the volatile anesthetic methoxyflurane but not the substrate benzphetamine [E. Canova-Davis and L. Waskell (1984) J. Biol. Chem. 259, 2541-2546]. To determine whether the requirement for cytochrome b5 for methoxyflurane oxidation is mediated by an allosteric effect on cytochrome P450 LM2 or cytochrome P450 reductase, we have investigated whether this anesthetic can induce a role for cytochrome b5 in benzphetamine metabolism. Using rabbit liver microsomes and antibodies raised in guinea pigs against rabbit cytochrome b5, we found that methoxyflurane did not create a cytochrome b5 requirement for benzphetamine metabolism. Methoxyflurane also failed to induce a role for cytochrome b5 in benzphetamine metabolism in the purified, reconstituted mixed function oxidase system. Studies of the reaction kinetics established that in the absence of cytochrome b5, methoxyflurane and benzphetamine are competitive inhibitors, and that in the presence of cytochrome b5, benzphetamine and methoxyflurane are two alternate substrates in competition for a single site on the same enzyme. These results all indicate that the methoxyflurane-induced cytochrome b5 dependence of the mixed function oxidase cytochrome P450 LM2 system is a direct result of the interaction between methoxyflurane and the substrate binding site of cytochrome P450 LM2 and suggest the focus of future studies of this question.     

Interaction of cholesterol hydroxylating cytochrome P-450 with cytochrome b5

Some new relations between cytochrome P-450-dependent monooxygenases were discovered. Cytochrome b5, a representative of "microsomal" monooxygenases, was shown to form a highly specific complex with cytochrome P-450scc, a member of the "ferredoxin" monooxygenase family. This interaction is characterized by a dissociation constant, Kd, of 0.28 microM. The cytochrome P-450scc-cytochrome b5 complex may be cross-linked with water-soluble carbodiimide. Using proteolytic modification of cytochrome b5, it was shown that both hydrophilic and hydrophobic fragments of cytochrome b5 are involved in the interaction with cytochrome P-450scc. Cytochrome b5 immobilized via amino groups is an effective affinity matrix for cytochrome P-450scc purification. The role of some amino acid residues in cytochrome P-450scc interaction with cytochrome b5 was studied. The role and the nature of complexes in cytochrome P-450-dependent monooxygenases as well as interrelationships between "microsomal" and "ferredoxin" monooxygenases are discussed.     

Mutant and immunochemical studies on the involvement of cytochrome b5 in fatty acid desaturation by yeast microsomes.

The involvement of cytochrome b5 in palmitoyl-CoA desaturation by yeast microsomes was studied by using yeast mutants requiring unsaturated fatty acids and an antibody to yeast cytochrome b5. The mutants used were an unsaturated fatty acid auxotroph (strain E5) and a pleiotropic mutant (strain Ole 3) which requires either Tween 80 and ergosterol or delta-aminolevulinic acid for growth. Microsomes from the wild-type strain possessed both the desaturase activity and cytochrome b5, whereas those from mutant E5 contained the cytochrome but lacked the desaturase activity. Microsomes from mutant Ole 3 grown with Tween 80 plus ergosterol were devoid of both the desaturase activity and cytochrome b5, but those from delta-aminolevulinic acid-grown mutant Ole 3 contained cytochrome b5 and catalyzed the desaturation. The cytochrome b5 content in microsomes from mutant Ole 3 could be varied by changing the delta-aminolevulinic acid concentration in the growth medium, and the desaturase activity of the microsomes increased as their cytochrome b5 content was increased. The antibody to yeast cytochrome b5, but not the control gamma-globulin fraction, inhibited the NADH-cytochrome c reductase and NADH-dependent desaturase activities of the wild-type microsomes. It is concluded that cytochrome b5 is actually involved in the desaturase system of yeast microsomes. The lack of desaturase activity in mutant Ole 3 grown with Tween 80 plus ergosterol seems to be due to the absence of cytochrome b5 in microsomes, whereas the genetic lesion in mutant E5 appears to be located at ther terminal desaturase.     

Chemical modification of cytochrome b5, cytochrome c and myoglobin with diethylpyrocarbonate

Cytochrome b5 is required for the cytochrome P-450 LM2 catalyzed oxidation of the anesthetic methoxyflurane. The ability of cytochrome b5 to support methoxyfluorane oxidation is affected by treatment with diethylpyrocarbonate, a reagent that at neutral pH is relatively specific for histidine residues. This inactivation of cytochrome b5 is reversed with hydroxylamine, which also suggests but does not prove histidine involvement. The studies reported in this paper were undertaken to determine whether histidine modification was involved in the decrease in effectiveness of cytochrome b5, or whether the inactivation could be attributed to modification of another amino acid. Our experiments demonstrate that diethylpyrocarbonate inactivates detergent-solubilized cytochrome b5 by modifying the axial histidines and displacing the heme. Because of the unexpected ease with which diethylpyrocarbonate displaced the heme from cytochrome b5, this same process was investigated in two other hemoproteins, cytochrome c and myoglobin. Diethylpyrocarbonate could not dissociate the heme from cytochrome c, whereas the heme was lost from myoglobin even more readily than from cytochrome b5.     

Responses of two protein-protein complexes to solvent stress: does water play a role at the interface?

We have analyzed the stability of the cytochrome c-cytochrome b5 and cytochrome c-cytochrome c oxidase complexes as a function of solvent stress. High concentrations of glycerol were used to displace the two equilibria. Glycerol promotes complex formation between cytochrome c and cytochrome b5 but inhibits that between cytochrome c and cytochrome c oxidase. The results with cytochrome b5 and cytochrome c were expected; the association of this complex is largely entropy driven. Our interpretation is that the cytochrome c-cytochrome b5 complex excludes water. The results with the cytochrome c oxidase and cytochrome c couple were not expected. We interpret them to mean that either glycerol is binding to the oxidase, thereby displacing the cytochrome c, or that water is required at this protein-protein interface. A requirement for substantial quantities of water at the interface of some protein complexes is logical but has been reported only once.     

The role of the hydrophobic fragment of cytochrome b5 in the interaction with cytochrome P-450

The interaction of highly purified liver microsomal cytochrome P-450 from phenobarbital-induced rabbits and cytochrome b5 has been investigated by the difference and second derivative difference spectroscopy. The addition of cytochrome b5 to cytochrome P-450 results in transition of cytochrome P-450 heme iron from low to high spin state. The interaction is accompanied by the changes in the second derivative spectrum of cytochrome P-450, which point to the participation of tryptophanyl residues in this process. The hydrophilic fragment of cytochrome b5 is unable to form a complex with cytochrome P-450 as judged by the absence of the difference spectrum and any changes in the second derivative UV-spectrum of cytochrome P-450. The evidence obtained indicates that the hydrophobic tail of the cytochrome b5 molecule responsible for its binding to membrane is also indispensable for forming a functional cytochrome P-450-cytochrome b5 complex.     

Genetic analysis of cytochrome b5 from arachidonic acid-producing fungus, Mortierella alpina 1S-4: cloning, RNA editing and expression of the gene in Escherichia coli, and purification and characterization of the gene product.

Information on the amino acid sequences of the internal peptide fragments of cytochrome b5 from Mortierella hygrophila was used to prepare synthetic oligonucleotides as primers for the polymerase chain reaction. A 100-base DNA fragment was thus amplified, by using a genomic gene from Mortierella alpina 1S-4 as a template, which produced polyunsaturated fatty acids such as arachidonic acid. The amplified DNA fragment was used as the probe to clone both a 523-base cDNA fragment and a 2.1-kilobase SalI-NruI genomic fragment coding for the whole M. alpina 1S-4 cytochrome b5. On the basis of nucleotide sequences of both cytochrome b5 genomic gene and cDNA, the genomic cytochrome b5 gene was found to consist of four exons and three introns. A novel type of RNA editing, in which the cDNA included either guanine insertion or adenine-->guanine substitution at one base upstream of poly(A), was interestingly observed. The deduced amino acid sequence of M. alpina 1S-4 cytochrome b5 showed significant similarities with those of cytochrome b5s from other organisms such as rat, chicken, and yeast. The soluble form of the cytochrome b5 gene was expressed to 16% of the total soluble protein in Escherichia coli. The holo-cytochrome b5 accounted for 8% of the total cytochrome b5 in the transformants. The purified cytochrome b5 showed the oxidized and reduced absorbance spectra characteristic of fungal microsomal cytochrome b5.     

Expression of outer mitochondrial membrane cytochrome b 5 in Escherichia coli. Purification of the recombinant protein and studies of its interaction with electron-transfer partners

In the present work, we report expression in Escherichia coli, purification, and characterization of recombinant full-length cytochrome b(5) from outer mitochondrial membrane. Optimization of expression conditions for cytochrome b(5) from outer mitochondrial membrane allowed reaching expression level up to 10(4) nmol of the hemeprotein per liter of culture. Recombinant cytochrome b(5) from outer mitochondrial membrane was purified from cell lysate by using metal-affinity chromatography. It has physicochemical, spectral, and immunochemical properties similar to those of cytochrome b(5) from rat liver outer mitochondrial membrane. Immobilized recombinant mitochondrial cytochrome b(5) was used as affinity ligand to study its interaction with electron transfer proteins. By using this approach, it is shown that in interaction of NADPH:cytochrome P450 reductase with both forms of cytochrome b(5) an important role is played by hydrophobic interactions between proteins, although the contribution of these interactions in complex formation with NADPH:cytochrome P450 reductase is different for isoforms of cytochrome b(5).     

The opposite effect of bivalent cations on cytochrome b5 reduction by NADH:cytochrome b5 reductase and NADPH:cytochrome c reductase.

The effects of bivalent cations on cytochrome b5 reduction by NADH:cytochrome b5 reductase and NADPH:cytochrome c reductase were studied with the proteinase-solubilized enzymes. Cytochrome b5 reduction by NADH:cytochrome b5 reductase was strongly inhibited by CaCl2 or MgCl2. When 1.2 microM-cytochrome b5 was used, the concentrations of CaCl2 and MgCl2 required for 50% inhibition (I50) were 8 and 18 mM respectively. The inhibition was competitive with respect to cytochrome b5. The extent of inhibition by CaCl2 or MgCl2 was much higher than that by KCl or other alkali halides. In contrast, cytochrome b5 reduction by NADPH:cytochrome c reductase was extremely activated by CaCl2 or MgCl2. In the presence of 5 mM-CaCl2, the activity was 24-fold higher than control when 4.4 microM-cytochrome b5 was used. The magnitude of activation by CaCl2 was 2-3-fold higher than that by MgCl2. The activation by these salts was much higher than that by KCl, indicating that bivalent cations play an important role in this activation. The mechanisms of inhibition and activation by bivalent cations of cytochrome b5 reduction by these two microsomal reductases are discussed.     

Interaction of coumarin-hydroxylating cytochrome P-450coh from liver microsomes of mice induced by pyrazole with cytochrome b5

Cytochrome P-450coh from pyrazole-treated mice was shown to form a tight and specific complex with cytochrome b5 from mouse liver microsomes. The complex formation was found to result in type I spectral changes indicating a spin shift from the low to the high spin form. When added to a reconstituted system containing cytochrome P-450coh, NADPH-cytochrome P-450 reductase and phospholipid, cytochrome b5 stimulates hydroxylation of coumarin and O-deethylation of 7-ethoxycoumarin. The maximal stimulating effect is reached at a 1:1 stoichiometry. Mouse liver cytochrome b5 stimulates hydroxylation and deethylation by 100% and 60%, respectively. The stimulating effect of cytochrome b5 was found to result from the increase of the maximal rate of oxidation, being practically without effect on Km. Cytochrome b5 purified from rat and rabbit liver microsomes interacts with cytochrome P-450coh but fails to stimulate the oxidation reaction. At large excess, cytochrome b5 inhibits the oxidations catalyzed by cytochrome P-450coh. Immobilized cytochrome b5 either from mouse or rat and rabbit microsomes proved to be an efficient affinity matrix for cytochrome P-450coh purification.     

Models for the complexes formed between cytochrome b5 and the subunits of methemoglobin

Computer graphics-generated models for the electron transfer complexes formed between cytochrome b5 and the subunits of methemoglobin are proposed. For both complexes, the orientation allowing optimal hydrogen bonding involves interaction between negatively charged residues on cytochrome b5 and positively charged residues on methemoglobin. In each complex, the heme groups of the interacting species are coplanar with the edges of the heme groups separated by 7-8 A and with the iron atoms 16 A apart. For the alpha-chain X cytochrome b5 complex, alpha-chain residues 56 (Lys), 60 (Lys), and 90 (Lys) interact with cytochrome b5 residues 44 (Glu), 43 (Glu), and 60 (Asp) respectively. A fourth hydrogen bond involves alpha-61 (Lys) bridging between a heme propionate from cytochrome b5 and a heme propionate from the alpha-chain. The contacts present in the beta-chain X cytochrome b5 complex involve hydrogen-bonding between beta-chain lysyl residues 59, 61, 65, and 95, and cytochrome b5 residues 48 (Glu), 44 (Glu), 43 (Glu), and 60 (Asp) respectively. An additional hydrogen bond can be formed by bridging of the epsilon-amino group of beta-66 (Lys) between a heme propionate from cytochrome b5 and a beta-chain heme propionate. In each complex, two nonionic interactions, one on each side of the heme groups, are also suggested. These interactions appear to effectively exclude external water molecules from the center of the protein-protein interaction domain. Comparison of the proposed binding loci for cytochrome b5 on the methemoglobin subunits with those proposed on cytochrome c reveals considerable structural homology between the cytochrome b5 binding sites.