THE JOURNAI. OF BIOLOGICAL CHEMISTRY Vol. 259, No. 4, Issue of February 25, pp. 2541-2546, 1984 Printed in U.S. A.

The Identification of the Heat-stable Microsomal Protein Required for Methoxyflurane Metabolism as Cytochrome b5*

(Received for publication, July 8, 1983)

Eleanor Canova-Davis and Lucy Waskells From the Department of Anesthesia, Veterans Administration Medical Center, San Francisco, California 94121 and Liver Center, University of California, S a n Francisco, California 94143

Methoxyflurane is an anesthetic whose metabolism by cytochrome P-45OLM2 has been shown to be depend- ent upon a heat-stable microsomal protein (Canova- Davis, E., and Waskell, L. A. (1982) Biochem. Bio- phys. Res. Commun. 108, 1264-1270). Treatment of this protein with diethylpyrocarbonate, which modi- fies selected amino acids, caused a dose-dependent loss in its ability to effect the metabolism of methoxyflur- ane by purified cytochrome P-450~~1,. This protein factor has been identified as cytochrome b6 by demon- strating that cytochrome b, and the heat-stable factor coelute during cytochrome bs purification. Neither fer- riheme nor apocytochrome b5 was able to substitute for the activating factor, while cytochrome bg reconsti- tuted from apocytoehrome b6 and heme exhibited an activity similar to that of native be. Examination of the cytochrome b, molecule by computer graphics sug- gested that diethylpyrocarbonate did not inactivate bs by reacting with the anionic surface of the cytochrome 6, molecule. Maximal rates of methoxyflurane metab- olism were obtained at a ratio of 1:l:l of the three proteins, cytochrome P-450LM,:reductase:cytochrome 6,. In summary, it has been demonstrated that the heat- stable protein, cytochrome b5, is obligatory for the metabolism of methoxyflurane by cytochrome P-450LM,. These data also suggest that cytochrome bs may be acting as an electron donor to P - 4 5 0 ~ ~ ~ in the 0-demethylation of methoxyflurane.

Methoxyflurane (2,2-dichloro-l,l-difluoroethyl methyl ether) is a volatile anesthetic whose biotransformation is effected in hepatic microsomes. It is readily 0-demethylated to formaldehyde, dichloroacetic acid, and fluoride ion which is nephrotoxic. The participation of cytochrome P-450 in this reaction is suggested by the following observations: 1) me- thoxyflurane produces a Type I difference spectrum when incubated with microsomes indicating t,hat the anesthetic binds to the monooxygenase (Takahashi et al., 1974); 2) its metabolism is inhibited by carbon monoxide (Ivanetich et al., 1979) and by antibodies to NADPH cytochrome P-450 reduc- tase (Waskell and Gonzales, 1982); and 3 ) its biotransforma- tion is induced 10-fold by phenobarbital (Greenstein et al., 1975).

In spite of the fact that methoxyflurane metabolism was

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

3 Recipient of Grant GM-27791 from the National Institutes of Health. To whom reprint requests and correspondence should be sent.

induced by phenobarbital, a purified preparation of the phen- obarbital-induced P-450, known as LMz, was unable to me- tabolize methoxyflurane.’ Hence, an effort was made to isolate the P-450 isozyme responsible for methoxyflurane metabo- lism by monitoring activity in addition to the customary tabulation of P-450 content. I t was discovered that although the methoxyflurane-metabolizing ability of P-450-containing fractions was lost after ion exchange chromatography, the activity could be restored by the addition of heat-treated polyethylene glycol-precipitated fractions of the solubilized microsomes (Canova-Davis and Waskell, 1982). The results presented herein demonstrate that cytochrome bs is the essen- tial component in these fractions and probably functions by transferring the second electron to P-450LM,.2

EXPERIMENTAL PROCEDURES

Purification of Microsomal Enzymes-Liver microsomes were pre- pared from phenobarbital-treated white New Zealand male rabbits as described by Haugen and Coon (1976). The procedure for obtaining the heat-stable factor from microsomes has been previously published (Canova-Davis and Waskell, 1982). The method of Dignam and Strobel (1975) as modified by Vermilion and Coon (1978) was followed for the isolation of NADPH-cytochrome P-450 reductase from pyro- phosphate-extracted phenobarbital-induced rabbit liver microsomes. The preparation was electrophoretically homogeneous and had a specific act,ivity of 51 pmol of cytochrome c reduced/min/mg of protein in 0.3 M potassium phosphate buffer, pH 7.7 a t 30 “C.

The purification to electrophoretic homogeneity of P-4501.M~ from phenobarbital-treated rabbits was performed essentially according to Johnson et al. (1979). Pyrophosphate-washed microsomes were solu- bilized with sodium cholate and fractionated with polyethylene glycol. A 6-10% polyethylene glycol fraction was obtained and applied to a DE52 cellulose column equilibrated with 10 mM Tris acetate, pH 7.4 a t 25 “C, 20% glycerol, 0.3% Nonidet P-40, and 1 mM EDTA (Buffer A). After the P-450LM2-containing fraction was eluted, a P-450L~,,- enriched fraction was obtained by eluting with 0.06 M KC1 in Buffer A. Increasing the KC1 content to 0.3 M released the remainder of the proteins from the column. The P-45O~M,-containing peak was treated with calcium phosphate gel and chromatographed on a CM52 cellulose column as described by Johnson et al. (1979) except that the Nonidet P-40 concentration was increased to 0.3%. The P-450LM,-containing peak from this CM52 cellulose column was treated with calcium phosphate gel and applied to an hydroxyapatite-agarose gel column equilibrated with 10 mM KPO,, pH 7.4, 20% glycerol, 0.1 mM EDTA, and 0.3% Nonidet P-40. The column was then washed with 3 column volumes of starting buffer and purified P-450LM, was eluted with 55 mM buffer. The other cytochromes were removed from the column with 200 mM buffer. The concentration of cytochrome P-450 was determined by the method of Omura and Sato (1964). The specific

L. A. Waskell, unpublished observation. The abbreviation used is: P-4501.~, liver microsomal cytochrome

P-450. The isozymes of rabbit liver microsomal cytochrome P-450 are numbered according to their relative electrophoretic mobilities, in accordance with the general recommendation of the Committee on Biochemical Nomenclature of the International Union of Biochem- istry.

2541

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2542 Role of Cytochrome b5 in Hydroxylations by Cytochrome P-450

content of the purified sample was 17.2 nmol of P-450/mg of protein. The partially purified P-450 used in some studies was obtained after the DE52 cellulose chromatography; specific activity: 7.2-7.9 nmol of P-450/mg of protein.

The method of Chiang (1981) was used for the purification of bg from detergent-solubilized microsomes of phenobarbital-treated rab- bits with the following modifications. The detergent-solubilized mi- crosomes were applied to a DE52 cellulose column equilibrated with 10 mM Tris-chloride, pH 7.7 a t 4 "C, 10% glycerol, 0.1 mM EDTA, and 0.4% Nonidet P-40. The hemeprotein fraction which eluted with 0.1 M KC1 in the equilibration buffer was reserved for the isolation of b5 and partially purified P-450LM2. The reductase was absorbed to the column and was purified as previously published (Vermilion and Coon, 1978). The hemeprotein fraction was applied to a DE52 cellu- lose column as described for the isolation of P-450LM2. The protein fraction which was eluted with 0.3 M KC1 was enriched in cytochrome bs. This fraction was further purified on DE52 cellulose and Sephadex G-100 columns as described by Chiang (1981). The concentration of purified b5 was ascertained from the absolute spectrum of the ferric protein using an absorption coefficient of 117 mM" cm" a t 413 nm (Strittmatter and Velick, 1956). In cruder preparations the amount of b5 was estimated from the reduced versus oxidized spectra (Esta- brook and Werringloer, 1978 Ozols, 1974).

Reconstitution of Apocytochrome bg with Ferriheme-Apocyto- chrome 6, was prepared by an acid-acetone treatment as described by Cinti and Ozols (1975) as modified by Vatsis et al. (1982). The reconstitution of b, was effected according to Vatsis et al. (1982) except that excess hemin was removed by dialysis through Spectra/ Por 6 membrane tubing (molecular weight cutoff, 3,500; Spectrum Medical Industries).

Benzphetamine N-Demethylation in the Reconstituted Enzyme Sys- tem-Reaction mixtures were prepared in a total volume of 300 p l of 0.05 M Tris acetate buffer, pH 7.4 at 37 "C, containing an NADPH- generating system consisting of 10 mM glucose 6-phosphate, 1 mM NADP, and 0.3 unit of glucose-6-phosphate dehydrogenase. The P-450LM, preparation was first combined with the reductase followed by 20 pg of dilauroylglyceryl-3-phosphorylcholine before addition of the aqueous components. The reaction was begun by the addition of benzphetamine to a final concentration of 1 mM. Incubation was conducted at 37 "C in a shaking water bath for 20 min. The reaction was terminated by the addition of 33 p l of 70% trichloroacetic acid. After centrifugation for 3 min in an Eppendorf microfuge the form- aldehyde content of the supernatant solution was determined.

Methoxyflurane 0-Demethylation in the Reconstituted Enzyme Sys- tem-Reaction mixtures were prepared in a total volume of 300 p1 of 0.1 M Tris acetate buffer, pH 7.4 a t 37 "C, containing an NADPH- generating system consisting of 10 mM glucose 6-phosphate, 1 mM NADP, and 0.3 unit of glucose-6-phosphate dehydrogenase. The P-450LM, preparation was first combined with the NADPH-P-450 reductase, followed by 20 pg of dilauroylglyceryl-3-phosphorylcholine and either b, or the heat-treated polyethyleneglycol fraction before addition of the aqueous components. When the heat-treated poly- ethyleneglycol fraction was used, the incubation mixture required Nonidet P-40 a t a final concentration of 0.1% presumably to aid in the solubilization of the fraction (Canova-Davis and Waskell, 1982). The reaction was begun by the addition of 1 11 of methoxyflurane. Incubation was conducted a t 37 "C in a shaking water bath for 30-60 min. The reaction was terminated by a 2-min treatment in a 75 "C water bath. After centrifugation for 3 min in an Eppendorf microfuge the fluoride ion content of the supernatant fluid was determined.

Other Methods-Inorganic fluoride was measured with an Orion ion-specific electrode, and formaldehyde was monitored as described by Nash (1953). Protein concentrations were measured according to Lowry et al. (1951) after precipitation of the proteins in the presence of trichloroacetic acid and sodium deoxycholate (Peterson, 1977). Synthetic lipids were suspended in water by sonication. Optical spectra were recorded a t room temperature with a Cary 219 spectro- photometer operated in the double beam mode. Polyacrylamide slab gel electrophoresis was carried out in the presence of sodium dodecyl sulfate with the discontinuous buffer system described by Laemmli (1970).

Materials-Nonidet P-40 (nonionic detergent), D-glucose 6-phos- phate (monosodium salt), glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides, and P-nicotinamide adenine dinucleotide phosphate (sodium salt) were purchased from Sigma. The ion ex- change resins DE52 and CM52 cellulose were obtained from What- man. Sephadex G-50 and G-100 were from Pharmacia Fine Chemi-

cals; Hydroxyapatite-Ultrogel from LKB-Produkter, and calcium phosphate gel from Bio-Rad. The synthetic lipid dilauroylglyceryl-3- phosphorylcholine was purchased from Calbiochem-Behring, and pol- yethylene glycol 6000 was from J. T . Baker Chemical Co. Diethylpyr- ocarbonate and hydroxylamine hydrochloride were obtained from Aldrich. Allylisopropylacetamide was a gift from Dr. W. E. Scott of Hoffmann-La Roche Inc.; benzphetamine was from Dr. H. Loh of the University of California at San Francisco.

RESULTS

Proteinaceous Character of the Heat-stable Microsomal Fac- tor-As reported previously (Canova-Davis and Waskell, 1982) incubation with trypsin destroyed the activity of the heat-stable factor. Further evidence that the factor was a protein was provided by incubating fractions containing the factor with increasing concentrations of diethylpyrocarbon- ate, which modifies selected amino acids in proteins (Oster- man-Golkar et al., 1974). A dose-dependent inactivation of the heat-stable factor was effected (Fig. 1). Restoration of the activating properties of the heat-treated polyethyleneglycol fraction occurred upon decarbethoxylation with hydroxyl- amine, suggesting the involvement of histidine, serine, thre- onine, or tyrosine in the unknown protein.

Specifically, calmodulin and bovine serum albumin were inactive. Microsomes isolated in the presence of fluoride ion metabolized methoxyflurane to the same extent as micro- somes isolated without fluoride ion present, suggesting that the necessary protein was probably not a kinase (Goodwin et al., 1982).

Copurification of the Protein-activating Factor with Cyto- chrome b5-Studies of P-450-mediated microsomal reactions have led to proposals that b5 can play a role in the transfer of the second electron in substrate oxidations (Hildebrandt and Estabrook, 1971). Hence, purification of b5 was undertaken to determine if the heat-stable factor was indeed b5. As the specific activity of b5 in the various steps increased, so did the activation properties of the same samples, suggesting that the protein-activating factor and b5 might be identical (Table I).

Role of the Heme Prosthetic Group of Cytochrome b, in P-450LM2-catalyzed Methoxyflurane 0-Demethylation-To rule out the possibility that the copurification of the activating protein and holocytochrome b5 was not merely coincidental,

DEP 2 4 6 8 IO 12 14 16 ImM) NH,OH,HCI 20 40 60 80 100 120 140 160(mM)

FIG. 1. Inhibition of the heat-treated polyethylene glycol fraction with diethylpyrocarbonate and subsequent reacti- vation with hydroxylamine hydrochloride. The heat-treated polyethylene glycol fraction (250 pg) was preincubated with the indicated concentrations of diethylpyrocarbonate (DEP) a t room temperature for 30 min. The assay was conducted as described under "Experimental Procedures": partially purified P - 4 5 0 ~ ~ , , 1.8 nmol/ml; reductase, 0.32 nmol/ml; incubation time: 60 min. Controls at each point consisted of identical components except the heat-treated pol- yethylene glycol fraction was added after the diethylpyrocarbonate and assayed immediately. Reactivation was accomplished by adding hydroxylamine hydro,:hloride at the concentrations indicated to the diethylpyrocarbonate preincubation mixture and further incubated a t room temperature for 30 min before assay.

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Role of Cytochrome b5 in Hydroxylations by Cytochrome P-450 2543 TABLE I

Copurification of bs and activation factor

Preparation Protein Cytochrome bJ tion“

rng nrnollmg nmol F-/ mg/min

Pyrophosphate-treated micro- 4465 1.4 0.9

First DE52 cellulose column 1214 3.6 19.4

Second DE52 cellulose column 221 6.6 55.6

Third DE52 cellulose column 74 12.9 92.0

First Sephadex G-100 column 19 33.0 579.3

Second Sephadex G-100 col- 11 49.2 712.2

somes

eluate: hemoprotein fraction

eluate: b5-enriched fraction

eluate: b, fraction

eluate

umn eluate ~~

a Assayedas described under “Experimental Procedures”: partially purified P-45OLMz, 2.7 nmol/ml; reductase, 0.32 nmol/ml. Incubation was conducted for 30 min. Activation is normalized to 1.0 nmol of P- 450/ml.

[b,] f i M

Role of cvtochrome b, versus apocytochrome b, in the metabolism of methoxyflurane. The assaywas conducted as described under “Experimental Procedures”: partially purified P - 4 5 0 ~ ~ , , 2.7 nmoI/ml; reductase, 0.25 nmol/ml; O ” 0 , apocyto- chrome b5; o“-o, reconstituted bg; X”-& cytochrome b,; incu- bation time, 30 min.

the heme group was removed from b5 and the activating properties of the apoprotein were determined. If b5 is the protein which activates methoxyflurane metabolism, its activ- ity would be expected to decrease dramatically upon removal of the heme. Ferriheme itself is unable to substitute for the activating factor. The apocytochrome b5 prepared from bs gave no absorption bands in the Soret region when examined at a 2 HM concentration. The activating properties of b5 were essentially lost upon removal of the heme (Fig. 2). The small amount of activity present could be due to contaminating bs undetectable in the spectrum. The activity could be com- pletely restored upon reconstitution of bs.

Optimization of Methoxyflurane Metabolism in a Reconsti- tuted System-Since investigations of substrate metabolism in reconstituted enzyme systems are usually performed with P-450 as the rate-limiting component, experiments were un- dertaken to establish optimal levels of the reductase, b5, and phospholipid. The reaction is absolutely dependent upon P - 4 5 0 ~ ~ , , reductase, and NADPH (data not shown). As can be seen from Fig. 3, metabolite formation increases with increasing reductase concentration up to a 1:1:1 molar ratio of reductase:b6:P-450LM2. The conversion of methoxyflurane to its metabolites is illustrated as a function of b5 concentra- tion in Fig. 4. The reaction displayed an apparent V,, at equimolar amounts of the enzymes. Fig. 5 shows that the

1 2 1 8

Y I I I I I I I I 0.4

o.8 / I .2 16

Molar Ratio, [Reductase] [P-450LM2]

FIG. 3. Methoxyflurane metabolism by P - 4 5 0 ~ ~ ~ as a func- tion of reductase concentration. The assay was performed as described under “Experimental Procedures”: partially purified P - 4 5 0 ~ ~ * , 0.9 nmol/ml; b5, 0.9 nmol/ml; reductase as indicated; incu- bation time, 30 min.

36-

32 -

28 - C

i 24-

.-

\

z 20- - \ 0

16- e-..

E ‘Y 4 I I I I I I I I I

0 2 0.6 1.0 I .4

Molar Rotia , [ b5]/[ P-450LM2]

FIG. 4. Methoxyflurane metabolism of P - 4 5 0 ~ ~ , as a func- tion of cytochrome ba concentration. The assay was performed as described under “Experimental Procedures”: partially purified P - 4 5 0 ~ ~ , 0.9 nmol/ml; reductase, 0.9 nmol/ml; b5 as indicated incu- bation time, 30 min.

activity in the absence of dilauroylglyceryl-3-phosphorylcho- line is approximately 25% of maximal, indicating a phospho- lipid dependence comparable in extent to that of other reac- tions catalyzed by P-450 in which b5 is not obligatory (Fasco et al., 1978). These results provide evidence for complex formation among the three proteins, although the product formation is not that anticipated for a simple 1:1:1 complex.

Comparison of the Metabolism of Methoxyflurane and Benz- phetamine by Cytochrome P-450LM2-The metabolism of both benzphetamine and methoxyflurane could be inhibited by allylisopropylacetamide. However, a sensitivity difference was observed. The concentration of inhibitor needed to inactivate

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2544 Role of Cytochrome b5 in Hydroxylations by Cytochrome P-450

20 60 100 140

Molar Ratio, [Dilauroyl-GPC]/[P-450~~~]

FIG. 5. Effect of dilauroylglyceryl-3-phosphorylcholine (ClilauroyZ-GPO concentration on methoxyflurane metabo- lism by P-450~~~. The assay was performed as described under “Experimental Procedures”: purified P-450LM,, 0.64 nmol/ml; b5, 0.64 nmol/ml; reductase, 0.64 nmol/ml; dilauroylglyceryl-3-phosphoryl- choline, as indicated; incubation time, 30 min.

TABLE I1 Inhibition of P-450LM2 by allylisopropylacetamide

150

Methoxyflurane Benzphetamine

m M

Preparation

Microsomes” 25 7.8 P-450?~, 25 7.8

Microsome concentration, 0.6 nmol of P-450/mI. Assayed as described under “Experimental Procedures”: partially

purified P -450~~* , 2.7 nmol/ml; reductase, 0.64 nmol/ml; incubation time, 30 min. The P-45oLM, concentration was reduced to 0.27 nmol/ ml for the metabolism of benzphetamine.

either the microsomal or reconstituted systems was lower for the metabolism of benzphetamine (Table 11). It was also possible to competitively inhibit the metabolism of benzphet- amine by methoxyflurane in both systems. In turn, benzphet- amine could competitively inhibit the metabolism of methox- yflurane. Both benzphetamine and methoxyflurane can bind to P-450 and induce a Type I spectrum. However, the maximal binding of benzphetamine was greater than that of methoxy- flurane in both the microsomal and the reconstituted systems (data not shown).

Examination of Cytochrome bs by Computer Graphics-The three-dimensional structure of the cytochrome bs molecule as viewed on the University of California at San Francisco Computer Graphics System3 showed that the amino acids histidine, serine, threonine, and tyrosine are not present on the anionic surface of the bs molecule (Fig. 6). This anionic surface of b, is composed of several carboxyl groups which surround the exposed heme edge (Salemme, 1976). These carboxyl groups on bs are involved in complementary charge pairing with basic groups in the proteins known to transfer electrons to and from bs (Ng et al., 1977; Gacon et al., 1980; Dailey and Strittmatter, 1979, 1980).

DISCUSSION

Besides demonstrating that the heat-stable factor was a protein, the experiments with diethylpyrocarbonate suggest that either a histidine, tyrosine, serine, or threonine residue

The anionic surface of the bs molecule shown in Fig. 6 was constructed and photographed at the Computer Graphics Laboratory by Dr. Judith McClarin. The laboratory is directed by Dr. Robert Langridge and supported by National Institutes of Health Grant RR1081.

on the bs molecule, which was subsequently identified as the heat-stable factor, is necessary for stimulating methoxyflur- ane degradation. This was deduced from the observation that hydroxylamine could restore activity to the factor. Modifica- tions on lysine or arginine residues or on a-amino groups are not reversed by treatment with hydroxylamine (Burstein et al., 1974). If diethylpyrocarbonate reacted with an amino acid residue on the anionic surface of b, and thus sterically inhib- ited its interaction with cationic groups on either P-4501,M, and/or P-450 reductase, electron transfer would be inhibited (Heinemann and Ozols, 1982; Dailey and Strittmatter, 1980). This possibility was eliminated by the examination of the three-dimensional structure of the bs molecule on the com- puter graphics system at the University of California at San Francisco (Fig. 6). Inspection of this anionic surface revealed that susceptible amino acids were not found on this face of the molecule. Three possible sites for the carbethoxylation by diethylpyrocarbonate exist. 1) The two ligands to the heme of bs are histidine residues (Argos and Mathews, 1975). 2 ) The hydrophobic tail of bs which is essential for electron transfer to the P-450 system (Chiang, 1981) contains suscep- tible residues (Dailey and Strittmatter, 1981). 3) Serine 64 (Fig. 61, which is immediately adjacent to this surface, may function in stabilizing the heme in its pocket (Argos and Mathews, 1975).

Since several laboratories (Vatsis et al., 1980; Kusunose et al., 1981; Okita et al., 1981) had reported that boiled bs was unable to stimulate P-450-mediated oxidation, the heat-stable factor was not initially considered to be bs. In addition Kita- gawa and co-workers (1982) reported that above 55 “C irre- versible denaturation of bs occurred. However, after publica- tion of the initial findings of the heat-stable factor (Canova- Davis and Waskell, 1982) it was learned that bs had to be boiled for several hours to significantly decrease i t s a~ t iv i ty .~ Consequently, the purification of bs was effected to determine if the heat-stable factor was indeed bs. As the specific activity of bs in the various steps increased, so did the activation properties (Table I), establishing their identity.

The role of b5 in the P-450-catalyzed oxidation of methox- yflurane was investigated in a number of ways. Removal of the heme prosthetic group, and the subsequent inactivity of the apocytochrome b, suggested that electron transfer had been disrupted. Recovery of activity was effected upon recon- stitution of b, by insertion of ferriheme into the apocyto- chrome bs molecule. The involvement of bs in a 1:l molar ratio with P-450LM, and the reductase (Figs. 3 and 4) supports its postulated role as the second electron donor in the metab- olism of methoxyflurane. The inhibition studies with allyli- sopropylacetamide (Table 11) indicated that different enzyme systems participate in the metabolism of benzphetamine as opposed to methoxyflurane. This latter observation probably reflects the requirement for the additional protein-protein interaction of bs with P-450 in the metabolism of methoxy- flurane.

The observed obligatory role for bs in the NADPH-depend- ent monooxygenation, of methoxyflurane with purified P-4501,M2 extends the list of substrates with such a require- ment. Cytochrome & has been found to be necessary in a number of systems (Vatsis et al., 1980, 1982; Kuwahara and Omura, 1980; Sugiyama et al., 1980). It appears that the involvement of bs in reconstituted drug oxidation systems varies according to the substrate being metabolized, even when the molecular species of P-450 is the same; e.g. note the difference in the requirement of b, in the metabolism of

M. J . Coon, personal communication.

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Role of Cytochrome b, in Hydroxylations by Cytochrome P-450 2545

I

II FIG. 6. A stereoscopic view of the anionic surface (in red) of the cytochrome bs molecule

methoxyflurane as compared to that of benzphetamine (Can- ova-Davis and Waskell, 1983). Furthermore, the metabolism of the same substrate by different forms of P-450 also exhibits different requirements for bg, as demonstrated by the work of Vatsis et al. (1982) with the prostaglandins. In addition to its obligatory role, bs has been implicated as a participant in the reconstituted P-450 system in a facilitory role (Imai and Sato, 1977; Ingelman-Sundberg and Johansson, 1980; Okita et al., 1981; Bosterling et al., 1982; Vatsis et al., 1982).

The results discussed above demonstrate that bs plays an important role in the biotransformation of the anesthetic methoxyflurane. The function of b5 in this reaction is presum- ably to transfer the second of the two electrons that are required for substrate oxidation (Bonfils et al., 1981). At the present time there is no satisfactory explanation for the biological function or mechanism of this phenomenon. The question of how the oxyferro-substrate complex of a presum- ably unique species of P-450 is able to accept the second electron from the reductase with one substrate and from b5 with another substrate is intriguing. One may envision that substrate-induced allosteric effects on a given P-450 can result in an altered affinity of the oxyferro-substrate complex for the reductase versus b6.

The in vivo role of bs is to provide electrons for a host of important biosynthetic reactions (Passon et al., 1972; Oshino et al., 1971; Okayasu et al., 1977; Reddy et al., 1977; Grinstead and Gaylor, 1982; Paltauf et al., 1974; Keyes et al., 1979; Strittmatter et al., 1982). The oxidation by P-450 of selected drugs and/or endogenous substrates which requires b6 com- petes for these electrons. Such oxidation reactions may shift the balance between the various pathways, with possible significant consequences on the biochemical homeostasis of an organism.

Acknowledgments-We thank Dr. Minor J. Coon and Dr. Eric F. Johnson for their helpful discussions and suggestions, and Dr. James R. Trudell and Dr. Minor J . Coon for their gift of purified cytochrome bs.

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E Canova-Davis and L Waskellmethoxyflurane metabolism as cytochrome b5.

The identification of the heat-stable microsomal protein required for

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