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.     

Hydroxylation of prostaglandins by inducible isozymes of rabbit liver microsomal cytochrome P-450. Participation of cytochrome b5

The hydroxylation of prostaglandin (PG) E1, PGE2, and PGA1 was investigated in a reconstituted rabbit liver microsomal enzyme system containing phenobarbital-inducible isozyme 2 or 5,6-benzoflavone-inducible isoenzyme 4 of P-450, NADPH-cytochrome P-450 reductase, phosphatidylcholine, and NADPH. Significant metabolism of prostaglandins by isozyme 2 occurred only in the presence of cytochrome b5. Under these conditions, PGE1 hydroxylation was linear with time (up to 45 min) and protein concentration, and maximal rates were obtained with a 1:1:2 molar ratio of reductase: cytochrome b5:P-450LM2. Moreover, P-450LM2 catalyzed the conversion of PGE1, PGE2, and PGA1 to the respective 19- and 20-hydroxy metabolites in a ratio of about 5:1, and displayed comparable activities toward the three prostaglandins based on the total products formed in 60 min. Apocytochrome b5 or ferriheme could not substitute for intact cytochrome b5, while reconstitution of apocytochrome b5 with ferriheme led to activities similar to those obtained with the native cytochrome. Isozyme 4 of P-450 differed markedly from isozyme 2 in that it catalyzed prostaglandin hydroxylation at substantial rates in the absence of cytochrome b5, was regiospecific for position 19 of all three prostaglandins, and had an order of activity of PGA1 greater than PGE1 greater than PGE2. P-450LM4 preparations from untreated and induced animals had similar activities with PGE1 and PGE2, respectively. Addition of cytochrome b5 resulted in a 20 to 30% increase in the rate of PGE1 hydroxylation and an appreciably greater enhancement in the extent of all the P-450LM4-catalyzed reactions, the stimulation being greatest with PGE2 (3-fold) and least with PGA1 (1.6-fold). Cytochrome b5 was thus required for maximal metabolism of all three prostaglandins, but did not alter the regiospecificity or the order of activity of P-450 isozyme 4 with the individual substrates. In the presence of cytochrome b5, the prostaglandin hydroxylase activities of isozyme 4 were two to six times higher than those of isozyme 2.     

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.     

The enhancement of cytochrome P-450 catalyzed methoxyflurane metabolism by a heat-stable microsomal protein

We report the existence of a microsomal, heat-stable, trypsin-sensitive factor that stimulates the O-demethylation of methoxyflurane (CHCl₂CF₂OCH₃) by partially purified preparations of rabbit hepatic cytochrome P-450. The factor is able to stimulate by five to twelve-fold the methoxyflurane metabolizing activity of cytochrome P-450. In contrast, the metabolism of benzphetamine is not affected by the presence of the factor. The factor is inactivated by extraction with methanol, chloroform, butanol and ethanol. It remains intact after treatment with 6M guanidine hydrochloride and is soluble in trifluoroethanol. Thus, the weight of evidence indicates that this factor is a rather hydrophobic protein.     

Properties of electrophoretically homogeneous phenobarbital-inducible and beta-naphthoflavone-inducible forms of liver microsomal cytochrome P-450.

Procedures are described for the isolation of two forms of rabbit liver microsomal liver microsomal cytochrome P-450 (P-450LM) in homogeneous state. They are designated by their relative electrophoretic mobilities on polyacrylamide gel in the presence of sodium dodecyl sulfate as P-450LM2 and P-450LM4. P-450LM2, which was isolated from phenobarbital-induced animals, has a subunit molecular weight of 48,700. The best preparations contain 20 nmol of the cytochrome per mg of protein and 1 molecule of heme per polypeptide chain. P-450LM4, which is induced by beta-naphthoflavone but is also present in phenobarbital-induced and untreated animals, was isolated from all three sources and found to have a subunit molecular weight of 55,300. The best preparations contain 17nmol of the cytochrome per mg of protein and 1 molecule of heme per polypeptide chain. Some of the purified preparations of the cytochromes, although electrophoretically homogeneous, contain apoenzyme due to heme loss during purification. The purified proteins contain no detectable NADPH-cytochrome P-450 reductase, cytochrome b5, or NADH-cytochrome b5 reductase, and only low levels of phospholipid (about 1 molecule per subunit). Amino acid analysis indicated that P-450LM2 and P-450LM4 are similar in composition, but the latter protein has about 60 additional residues. The COOH-terminal amino acid of P-450LM2 is arginine, as shown by carboxypeptidase treatment, whereas that of P-450LM4 is lysine. NH2-terminal amino acid residues could not be detected. Carbohydrate analysis indicated that both cytochromes contain 1 residue of glucosamine and 2 of mannose per polypeptide subunit. The optical spectra of the oxidized and reduced cytochromes and carbon monoxide complexes were determined. Oxidized P-450LM2 has maxima at 568, 535, and 418 nm characteristic of a low spin hemeprotein, and P450LM4 from beta-naphthoflavone-induced, phenobarbital-induced, or control microsomes has maxima at 645 and 394 nm, characteristic of the high spin state. The spectrum of -450lm4 becomes similar to that of P-450LM2 at high protein concentrations or upon the addition of detergent (Renex), whereas the spectrum of P-450LM2 is unaffected by the protein concentration or the presence of detergent. Electron paramagnetic resonance spectrometry of the purified cytochromes indicated that oxidized -450lm2 is in the low spin state, whereas P-450LM4 is largely, but not entirely, in the high spin state.     

Specificity in the activation and inhibition by flavonoids of benzo[a]pyrene hydroxylation by cytochrome P-450 isozymes from rabbit liver microsomes

The effect of flavone and 7,8-benzoflavone on the metabolism of benzo[a]pyrene to fluorescent phenols by five cytochrome P-450 isozymes obtained from rabbit liver microsomes was determined. Benzo[a]pyrene metabolism was stimulated more than 5-fold by the addition of 600 microM flavone to a reconstituted monooxygenase system consisting of NADPH, cytochrome P-450 reductase, dilauroylphosphatidylcholine, and cytochrome P-450LM3c or cytochrome P-450LM4. In contrast, an inhibitory effect of flavone on benzo[a]pyrene metabolism was observed when cytochrome P-450LM2, cytochrome P-450LM3b, or cytochrome P-450LM6 was used in the reconstituted system. 7,8-Benzoflavone (50-100 microM) stimulated benzo[a]pyrene metabolism by the reconstituted monooxygenase system about 10-fold when cytochrome P-450LM3c was used, but benzo[a]pyrene hydroxylation was strongly inhibited when 7,8-benzoflavone was added to the cytochrome P-450LM6-dependent system. Smaller effects of 7,8-benzoflavone were observed on the metabolism of benzo[a]pyrene by the cytochrome P-450LM2-, cytochrome P-450LM3b-, and cytochrome P-450LM4-dependent monooxygenase systems. These results demonstrate that the activating and inhibiting effects of flavone and 7,8-benzoflavone on benzo[a]pyrene metabolism depend on the type of cytochrome P-450 used in the reconstituted monooxygenase system.     

Acetone-inducible cytochrome P-450: purification, catalytic activity, and interaction with cytochrome b5

A procedure was developed for the purification of an acetone-inducible form of cytochrome P-450 (P-450ac) to electrophoretical homogeneity from liver microsomes of acetone-treated rats. The P-450ac preparation containing 16.0 to 16.5 nmol P-450/mg protein moved as a single protein band with an estimated molecular weight of 52,000 upon gel electrophoresis in the presence of sodium dodecyl sulfate. The ferric P-450ac showed an absorption maximum at 394 nm at 25 degrees C, suggesting that it exists mainly in the high-spin form. It also existed in the low-spin form, especially at lower temperatures, as indicated by the absorption maximum in the 412-nm region. Upon reconstitution with NADPH: cytochrome P-450 reductase and phospholipid, P-450ac efficiently catalyzed both the demethylation and denitrosation of N-nitrosodimethylamine (NDMA) showing Vmax values of 23.8 and 2.3 nmol min-1 nmol P-450-1, respectively. The catalytic activity of P-450ac was greatly affected by cytochrome b5 which decreased the Km values of these reactions by a factor of 10 and increased the Vmax values. Cytochrome b5 appeared to interact with P-450 at a molar ratio of 1:1 and an intact cytochrome b5 structure was required for such interaction. Among the substrates studied, the demethylation of NDMA was affected the most by cytochrome b5 and showed the highest rate. P-450ac also catalyzed the oxygenation of N-nitrosomethylethylamine and aniline and the activity was enhanced slightly by cytochrome b5. Cytochrome b5 did not enhance the P-450ac-catalyzed metabolism of other drug substrates such as benzphetamine, aminopyrine, and ethylmorphine. P-450ac appeared to be similar in property to the previously studied rat P-450et (ethanol-inducible), rat P-450j (isoniazid-inducible), and rabbit P-450LM3a (ethanol-inducible). These P-450 species represent a new class of P-450 isozymes that are important in the metabolism of many endobiotics and xenobiotics.     

Inverse relationship between cytochrome P-450 phosphorylation and complexation with cytochrome b5

Cytochrome P-450 LM2 purified from rabbit liver microsomes has been shown to be a substrate for cAMP-dependent protein kinase. Cytochrome b5, in contrast, was a very poor substrate for cAMP-dependent protein kinase, although it stimulated the activity of the kinase toward histone. When purified rabbit cytochrome b5 was mixed with purified LM2, phosphorylation of LM2 by cAMP-dependent protein kinase was inhibited approximately 80-90%. Recently, a functional covalent complex of cytochrome b5 and LM2 was prepared and purified to homogeneity (P.P. Tamburini and J.B. Schenkman (1987) Proc. Natl. Acad. Sci. USA 84, 11-15). When present as a covalent complex with cytochrome b5, the phosphorylation of LM2 in the complex by cAMP-dependent protein kinase was also inhibited about 80-90% relative to an equivalent amount of LM2 alone. On the other hand, when the LM2 was phosphorylated prior to interaction with cytochrome b5, the ability of the latter to perturb the spin equilibrium of LM2 and oxidation of p-nitroanisole by the LM2 was diminished to an extent comparable to the degree of phosphorylation. The results suggest either that the phosphorylation site on LM2 may be within the cytochrome b5 binding site or that phosphorylation and cytochrome b5 cause mutually exclusive conformational changes in LM2. In addition, eight different forms of cytochrome P-450 from the rat (RLM2, RLM3, fRLM4, RLM5, RLM5a, RLM5b, RLM6, and PBRLM5) were examined as potential substrates for cAMP-dependent protein kinase under the same conditions. Maximal phosphorylation of about 20 mol% was obtained with LM2, and about half as much with PBRLM5. The low extent of phosphorylation of LM2 was not due to the prior presence of phosphate on the enzyme since LM2, as isolated, contains less than 0.1 mol phosphate/mol of enzyme. The other forms of cytochrome P-450 tested showed little or no phosphorylation in vitro despite the presence of a cAMP-dependent protein kinase phosphorylation sequence on at least two of them.     

Isoenzyme-specific phosphorylation of cytochromes P-450 and other drug metabolizing enzymes

A series of fourteen cytochrome P-450 isoenzymes was treated with three different protein kinases and found to divide into isoenzymes phosphorylated by both the cyclic AMP-dependent kinase and the calcium-phospholipid-dependent kinase (P-450 PB 3a and PB 2e), by none of these kinases (P-450 PB 1b, MC 1b, UT 1, and thromboxane synthase), and by either the cyclic AMP-dependent kinase (P-450 LM 2, PB 2d, and PB 3b) or the calcium-phospholipid-dependent kinase (P-450 PB 1a, PB 2a, MC 1a, LM 3c, and LM 4). Other components of the monooxygenase system, cytochrome P-450 reductase, cytochrome b5, cytochrome b5 reductase as well as microsomal epoxide hydrolase, were poor substrates for the kinases employed. On the other hand, glutathione transferases 1-2 and 4-4, but not 3-3, were relatively good substrates for the calcium-phospholipid-dependent kinase.     

Role of cytochrome b5 in catalysis by cytochrome P450 2B4

Cytochrome b5 has been shown to stimulate, inhibit or have no effect on catalysis by P450 cytochromes. Its action is known to depend on the isozyme of cytochrome P450, the substrate, and experimental conditions. Cytochrome P450 2B4 (CYP 2B4) has been used in our laboratory as a model isozyme to study the role of cytochrome b5 in cytochrome P450 catalysis using two substrates, methoxyflurane and benzphetamine. One substrate is the volatile anesthetic, methoxyflurane, whose metabolism is consistently markedly stimulated by cytochrome b5. The other is benzphetamine, whose metabolism is minimally modified by cytochrome b5. Determination of the stoichiometry of the metabolism of both substrates showed that the amount of product formed is the net result of the simultaneous stimulatory and inhibitory actions of cytochrome b5 on catalysis. Site-directed mutagenesis studies revealed that both cytochrome b5 and cytochrome P450 reductase interact with cytochrome P450 on its proximal surface on overlapping but non-identical binding sites. Comparison of the rate of reduction of oxyferrous CYP 2B4 and the rate of substrate oxidation by cyt b5 and reductase with stopped-flow spectrophotometric and rapid chemical quench experiments has demonstrated that although cytochrome b5 and reductase reduce oxyferrous CYP 2B4 at the same rate, substrate oxidation proceeds more slowly in the presence of the reductase.     

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.     

Modification of cytochrome P-450 with fluorescein isothiocyanate

Fluorescein isothiocyanate (FITC) has been shown to be selectively attached to the N-terminus of cytochrome P-450 LM2. The N-demethylase activity of cytochrome P-450 LM2 reconstituted systems modified in this way was inhibited by 25%. As revealed by CD measurements the overall conformation as well as the immediate heme environment of cytochrome P-450 LM2 remained unchanged after attachment of the FITC molecule. The binding affinity of modified cytochrome P-450 LM2 toward benzphetamine and aniline and the cumene hydroperoxide- or H2O2-supported N-demethylation of benzphetamine are maintained. However, the introduction of the electron via NADPH-cytochrome P-450 reductase (EC 1.6.2.4) is impaired after modification of the alpha-amino group. The extent of reduced modified cytochrome P-450 LM2 in the cytochrome P-450 reductase-supported reduction reaction is diminished and the half-time of the reduction is increased. The diminished reducibility is ascribed to steric hindrance of groups directly involved in the interaction between cytochrome P-450 LM2 and NADPH-cytochrome P-450 reductase or to blocking of the charge-pair interactions between the alpha-amino group of P-450 LM2 and the respective negatively charged group of NADPH-cytochrome P-450 reductase. By energy-transfer measurements distances between the heme and the alpha-amino group of 2.65 and 3.97 nm for the oligomeric and the monomeric forms of P-450 LM2, respectively, have been determined.     

Evidence of binary complex formations between cytochrome P-450, cytochrome b5, and NADPH-cytochrome P-450 reductase of hepatic microsomes

Water-soluble carbodiimide-catalyzed cross-linking of purified cytochrome P-450 LM2, cytochrome b5, and NADPH-cytochrome P-450 reductase was used to identify stable complexes formed between these proteins. High yields of P-450-b5 and P-450 reductase-b5 dimers, and lower yields of P-450 reductase-LM2 dimers were obtained. Substitution of native b5 and P-450 reductase with fully amidinated derivatives showed that LM2 and b5 were cross-linked exclusively through their respective amino and carboxyl groups. However, there appeared to be two complexation sites on the reductase which cross-link to b5 through amino groups and to LM2 through carboxyl groups respectively. A heterotrimer could not be identified following incubation of all three proteins together with EDC.     

Effects of hydrogen peroxide on cytochrome P-450 inactivation

Inactivation rate of purified oligomeric cytochrome P-450 LM2 has been investigated in glucose oxidase system and under the action of exogenous hydrogen peroxide (400 microM). It has been found that hydrogen peroxide has a distinct inactivating effect on cytochrome P-450. The enzyme inactivation is accompanied by the loss of heme and the decrease in SH-group content in the protein molecule. Benzphetamine, a substrate specific for this enzyme isoform, exerts a protective effect by decreasing the rate of cytochrome P-450 inactivation and SH-group oxidation. Similar results have been obtained during the investigation of cytochrome P-450 inactivation in the monomerized system. It has been found that the inactivation process is accompanied by the formation of the enzyme aggregates. The changes in the aggregate state are due to the formation of intermolecular covalent bonds.     

Degradation of rat hepatic cytochrome P-450 heme by 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine to irreversibly bound protein adducts

Administration of 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine (DDEP) (a structural analog of the dihydropyridine Ca2+ antagonists) to untreated, phenobarbital-, or dexamethasone-pretreated rats results in time-dependent losses of hepatic cytochrome P-450 content. Functional markers for various cytochrome P-450 isozymes have permitted the identification of P-450h, P-450 PB-1/k, and P-450p as the isozymes inactivated preferentially by the drug. DDEP-mediated cytochrome P-450 destruction may be reproduced in vitro, is most prominent after pretreatment of rats with dexamethasone, pregnenolone 16 alpha-carbonitrile or phenobarbital, and is blocked by triacetyloleandomycin. These findings together with the observation that DDEP markedly inactivates hepatic 2 beta- and 6 beta-testosterone hydroxylase and erythromycin N-demethylase tend to indict the steroid-inducible P-450p isozyme as a key protagonist in this event. The precise mechanism of such DDEP-mediated P-450p heme destruction is unclear, but involves prosthetic heme alkylation of the apocytochrome at its active site in what appears to be a novel mechanism-based "suicide" inactivation. Such inactivation appears to involve fragmentation of the heme to reactive metabolites that irreversibly bind to the protein, but the chemical structure of the heme-protein adducts is yet to be established. Intriguingly, such DDEP-mediated P-450p destruction in vivo also results in accelerated loss of immunochemically detectable apocytochrome P-450p. It remains to be determined whether or not this loss is due to enhanced proteolysis triggered by the structural modification of the apocytochrome.     

The study of the active site of cytochrome P-450 LM2 using the chemical modification of tyrosine residues by tetranitromethane

Selective chemical modification of the hemoprotein by tetranitromethane was used in order to elucidate the functional role of tyrosine residues in the cytochrome P-450 LM2 molecule. It was shown that the degree of cytochrome P-450 LM2 modification can be determined, using the second derivative of the UV absorption spectra. Modification of one tyrosine residue resulted in the inactivation of cytochrome P-450 LM2. Nitration of the cytochrome was accompanied by changes in the spectral properties of the hemoprotein with the formation of spectra typical of hyperporphyrin structures, thus suggesting the involvement of tyrosine residues in the formation of the active center of cytochrome P-450 LM2.     

Purification and characterization of a new form of cytochrome P-450 (LM2b) from untreated male rabbit liver microsomes

A new form of cytochrome P-450 has been purified from untreated male rabbit liver microsomes. This form was designated P-450 LM2b on the basis of its electrophoretic mobility on SDS polyacrylamide gel, where it migrates as a polypeptide of apparent molecular weight of 50,250. This hemoprotein exhibits a maximum at 448.5 nm in the Soret band of the CO-Ferrous state spectrum. On the basis of its molecular, spectral, enzymologic and immunologic data, P-450 LM2b was shown to be distinct from the other P-450 forms, already characterized in rabbit liver microsomes. However P-450 LM2b and P-450 LM3b appear to be immunologically related proteins.     

Benzo(a)pyrene metabolism by purified forms of rabbit liver microsomal cytochrome P-450, cytochrome b5 and epoxide hydrase in reconstituted phospholipid vesicles

The roles of rabbit liver cytochrome b₅, epoxide hydrase and various forms of cytochrome P-450 in the NADPH-dependent metabolism of benzo(a)pyrene were examined. After incorporation of the purified enzymes into phospholipid vesicles, using the cholate gel filtration technique, the various types of cytochrome P-450 did exhibit different stereospecificities in the oxygenation of the substrate. Cytochrome P-450_LM2 was found to efficiently convert benzo(a)pyrene in the presence of epoxide hydrase to 4,5-dihydroxy-4,5-dihydrobenzo(a)pyrene whereas cytochrome P-450_LM4 primarily participated in the formation of 9,10-dihydroxy-9,10-dihydrobenzo(a)pyrene. By contrast, benzo(a)pyrene was not metabolized by cytochrome P-450_LM3. Cytochrome b₅ enhanced cytochrome P-450_LM2-catalyzed oxygenations 5-fold, whereas cytochrome P-450_LM4-dependent oxygenations proceeded at a 3 times higher rate when cytochrome b₅ was present in the membrane.     

On the mechanism of action of cytochrome P-450. Oxidation and reduction of the ferrous dioxygen complex of liver microsomal cytochrome P-450 by cytochrome b5

The effects of cytochrome b5 on the decay of the ferrous dioxygen complexes of P-450LM2 and P-450LM4 from rabbit liver microsomes were studied by stopped-flow spectrophotometry. The P-450 (FeIIO2) complexes accept an electron from reduced cytochrome b5 and, in a reaction not previously described, donate an electron to oxidized cytochrome b5 to give ferric P-450. A comparison with the electron-transferring properties of ferrous P-450 under anaerobic conditions allowed determination of the limiting steps of the two reactions involving the oxygenated complex. The rate of decay of the dioxygen complex was increased in all cases with b5 present; however, with oxidized b5 a large increase in the rate was observed with P-450 isozyme 4 but not with isozyme 2, whereas the opposite situation was found when reduced b5 was used. The reactions between b5 and ferrous dioxygen P-450 were not at thermodynamic equilibrium under the conditions employed. From the results obtained, a model is proposed in which the ferrous dioxygen complex decomposes rapidly into another species differing from ferric P-450 in its spectral properties and from the starting complex in its electron-transferring properties. A scheme is presented to indicate how competition among spontaneous decay, cytochrome b5 oxidation, and cytochrome b5 reduction by the ferrous O2 complex may influence substrate hydroxylation.     

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.