ethyl isocyanide complexes of bacterial cytochrome p-450

9RCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 145, 220-229 (1971)

Ethyl lsocyanide Complexes of Bacterial Cytochrome P-450’

BRENDA GRIFFIN2 AND JULIAN A. PETERSON

The Department of Biochemistry, The University of Texas Southwestern Medical School at Dallas,

Dallas, Texas 75235

Received March 1, 1971; accepted April 20, 1971

The spectral properties of the ethyl isocyanide complexes of the soluble, highly purified cytochrome P-450, isolated from Pseudomonas putida, are reported. In the

presence of both camphor and potassiumions, the ethyl isocyanide complex of reduced bacterial cytochrome P-450 exhibits a single Soret absorption maximum at 453 nm, and

the magnitude of this maximum is unaffected by changes in pH between 6.0 and 8.0. The Soret absorption maximum of the ethyl isocyanide complex of reduced bacterial cytochrome P-420 is at 432 nm; an absorption maximum in the 430 nm region is ob-

served with bacterial cytochrome P-450 only under conditions which promote its conversion to a type of cytochrome P-420.

As a ligand of the heme iron of bacterial cytochrome P-450, ethyl isocyanide binds more tightly to the ferrous state than the ferric state, and the “apparent” K, of

ferrous cytochrome P-450 for ethyl isocyanide is 9.0 X 1OV M in the absence of camphor. The finding that camphor competes very effectively with the binding of ethyl

isocyanide to reduced cytochrome P-450 suggests that the camphor binding site is near the heme iron.

The hemoprotein, cytochrome P-450, has been shown by its photochemical action spectrum to be involved in t,he monooxygenation of various st’eroids, drugs, and xeno- biotics (1, 2). Considerable speculation has arisen regarding the active site of liver microsomal cytochrome P-450 because of the relative nonspecificity of this enzyme for the substances which it. hydroxylates (3). Omura and Sato (4, 5) showed that the complex formed between reduced microsomal cytochrome P-450 and ethyl isocyanide displays a Soret absorption maximum at 430 nm which is typical of hemoglobin (7), but the derivative of cytochrome P-450, unlike hemoglobin, has an additional maximum at 455 nm. The relative intensity of these ab-

r Supported, in part, by USPHS Research Grant AM-13366, from the Institute of Arthritis and Metabolic Diseases, and Research Grant I-405

from The Robert A. Welch Foundation, Houston, Texas.

2 Postdoctoral Fellow of The Robert A. Welch Foundation.

sorption maxima is sensitive to changes in pH and ionic strength of the buffer medium. At pH 6.0, the 430 nm absorption maximum is predominant,, but as the pH is increased the 455 nm absorption maximum increases at the expense of the 430 nm absorption maximum (8). In addition, at a given pH, the 455 nm absorption maximum is intensi- fied at the expense of the 430 nm absorption maximum by increasing the ionic st’rength of the buffer (9). In a study utilizing other alkyl isocyanides and phenyl isocyanide, Ichikawa and Yamano (10) showed that the effect on the two absorption maxima of increasing the hydrocarbon chain length of the isocyanide is qualitatively similar to increasing the ionic strength. It has been sug- gested that the 430 nm and 455 nm absorption maxima represent two different ethyl isocyanide complexes of reduced cytjochrome P-450, which exist in an equilibrium (8). The reduced state of cytochrome P-420, a denatured form of cytochrome P-450, also forms a complex with ethyl isocyanide, and

220

BACTERIAL CYTOCHROME P-450 221

the absorption spectrum has a single Soret’ maximum at, 433 nm (4).

A soluble bacterial cytochrome P-450, which was isolated from camphor-grown Pseudomonas putida by Katagiri et al. (11)) provides an excellent model for the study of monooxygenation reactions (12, 13). This bacterial cytochrome P-450 has been crystal- lized and several of it’s chemical and physical properties have been report,ed (14-17). The purpose of this study was : (1) to characterize the ethyl isocyanide complexes of the purified bacterial cytochrome P-450, and (2) to com- pare the absorption spectrum of the complex of the reduced form of bacterial cytochrome P-450 wit#h that of the corresponding complex of microsomal cytochrome P-450 in an at- tempt t,o understand better the origin of t’he 430 nm and 455 nm absorption maxima observed with microsomal cytochrome P-450.

Katagiri et al. (11) have designated their preparation of bacterial cytochrome P-450 as “P-450,,,.” To avoid confusion about t)he presence or absence of camphor in a given preparation of bacterial cytochrome P-450, we will not adopt t,heir t,erminology but, will refer, hereafter in this report, to bacterial cytochrome P-450 as “cytochrome P-450.” In the discussion, the bact,erial cytochrome P-450 will be differentiated from mammalian cytochromes P-450 by prefacing the names of the mammalian enzymes by their source,

e.g., “liver microsomal cyt’ochrome P-450” or “mammalian cytochrome P-4.50.”

MATERIALS AN11 METHOI)S

The isolation of cytochrome P-450 from cam-

phor-grown Pseudomonas puiida and the purifica- tion procedure are described in detail elsewhere (17). The ethyl isocyanide was a kind gift, of

Mr. Don Shoeman, Department of Pharmacology, University of Minnesota.

The absorption spectra were recorded with an

Aminco-Chance dual wavelength/split-beam re- cording spectrophotometer in the split-beam mode. A Varinn E4 EPR spectrometer with the variable

temperature apparatus was employed to record the EPR spectra.

RESULTS

The absolute absorption spectrum of t,he ethyl isocyanide complex of reduced cytochrome P-450, shown in Fig. 1, has a single absorption maximum in the Soret, region at

WAVELENGTH (nm)

FIG. 1. The effect of ethyl isocyanide on the absorption spectrum of reduced cytochrome P-450

in t,he presence of camphor. The reference cuvette contained 20 mM Tris-chloride buffer, pH 7.4, and

0.1 M KCl. The sample cuvette cont’ained the buffer solution which was 2.5 PM in cytochrome P-450

and 41 PM in camphor (- - -). Reduction of cytochrome P-450 was carried out by the addition of sodium dithionite (. . . . . . . ), after which ethyl

isocyanide was added to both cuvettes to a final concentration of 8.4 mM (p), resulting in a dilu-

tion of 107;, then carbon monoxide was bltbbled into t.he sample cuvette (-A-). Because the purity of the ethyl isocyanide was not determined,

the ethyl isocyanide concentrations may be overestimated.

453 nm (EM = 104 X lo3 I\I-’ cm-l) and appears to be completely free of the 430 nm absorption maximum which is typically observed with microsomal cytochrome P-450. Carbon monoxide, which binds strongly to reduced cyt,ochrome P-450, can compete with ethyl isocyanide for a ligand position of the heme iron (4, 1s). After the reaction mixture (Fig. 1) was saturat’ed with carbon monoxide, the absorption spectrum of the solution was identical to that of the carbon monoxide complex of reduced cytochrome P-450 which has a Soret absorption maximum at 447 nm.

Cytochrome P-450 is a very stable protein, and in the presence of camphor and K+ it can be stored for at least 4 weeks at 4” without formation of measurable amounts of cytochrome P-420 (17). However, in the absence of these substances cytochrome P-4.50 is degraded to a form of cytochrome P-420. Shown in Fig. 2 is the absorption spectrum of cytochrome P-420 prepared by storing for 1 week at, 4” a sample of cytochrome P-450 from which camphor and K+

222 GRIFFIN AND PETERSON

I I I I - Olldlzed

--Reduced

Reduced + CO 432

WAVELENGTH (nm)

FIG. 2. The effect of ethyl isocyanide on the absorption spectrum of reduced cytochrome

P-420. Cytochrome P-420 was formed from cytochrome P-450 as described in the text. Total hemoprotein concentration in the sample cuvette was approximately 6.5 PM in 0.1 M Tris-

chloride buffer, pH 7.4, and the reference cuvette contained the buffer; (--), oxidized cytochrome P-420; (- -), dithionite-reduced cytochrome P-420. The absorption spectra

of the carbon monoxide (. . . . . . ) and ethyl isocyanide (-A-) complexes of the reduced form were determined oh separate samples. Ethyl isocyanide concentration in both the

sample and reference cuvettes was 9.8 mM. The absorption spectrum of the ethyl isocyanide complex was virtually unchanged when carbon monoxide was bubbled into the sample cu-

vette.

had been removed. The Soret absorption maximum of the carbon monoxide complex of reduced cytochrome P-420 is at’ 421 nm.

The absorption spectrum of the ethyl isocyanide complex of reduced cytochrome P-420 has a maximum at 432 nm; the small shoulder at 453 nm is probably due t,o the presence of some cytochrome P-450 in the preparation. The absorption spectrum of the ethyl isocyanide complex of reduced cytochrome P-420 was essentially unchanged when carbon monoxide was bubbled into the solution.

Phenol is one of many substances known t,o convert microsomal cytochrome P-450 into an inactive form designated as micro-

somal cytochrome P-420 (19). Therefore, the reduction of cytochrome P-450 and subse- quent formation of the ethyl isocyanide complex were carried out, in a 1% phenolic buffer. When the difference absorption spectrum was monitored with time, the absorp-

tion maximum at 453 nm was found to decrease slowly while the maximum at 434 nm increased concomitantly. In an analogous experiment in which the ethyl isocyanide

was replaced with carbon monoxide, it was observed that the 447 nm absorption max-

imum slowly disappeared as the 421 nm absorption maximum, characteristic of cytochrome P-420, formed. These experiments indicate that the presence of the 434 nm absorption maximum of cytochrome P-450 is a measure of the amount of degraded cytochrome P-450, i.e., cytochrome P-420, present in the reaction mixture.

The changes in the absorption spectrum of microsomal cytochrome P-450 are usually investigated by means of difference spectro- photometry, due to the particulate nature of microsomal preparations. Therefore, the following experiments using the bacterial cytochrome P-450 were obtained as difference spectra to emphasize small changes in the absolute absorption spectrum and so these spectra would be directly comparable with t’he results previously obtained with mammalian cytochrome P-450. The difference spectrum of the ethyl isocyanide- reduced cytochrome P-450 complex relative to reduced cytochrome P-450 is shown in Fig. 3A to have an absorption maximum at 453 nm. In this experiment, both camphor and K+ ions were present, and the pH of the reaction mixture was 5.95, a pH at which the absorption maximum of the ethyl isocyanide


WAVELENGTH (nm)

FIG. 3. The effect of camphor and potassium ions on the difference spectrum of the ethyl isocyanide complex of reduced cytochrome P-450. In A, B, and C, the spectrum of the ethyl isocyanide complex of reduced cytochrome P-450 was recorded (-), and then carbon monoxide was bubbled into the sample cuvette (-.-). A. In the presence of both camphor and K+ both cuvettes contained 0.73 pM cytochrome P-450 and 34 ,uM camphor in 0.1 M potassium phosphate buffer, pH 5.95, to which sodium dithionite had been added. Ethyl isocyanide was added to the sample cuvette to a final concentration of 1.8 mM. B. In the absence of camphor and the presence of K+ both cuvettes contained 0.1 M Tris-chio- ride buffer, pH 5.95, 0.1 M KCl, 0.56 PM cytochrome P-450, and sodium dithionite. Ethyl isocyanide was added to the sample cuvette to a final concentration of 2.1 m&f. C. In the absence of both camphor and K+ conditions were those of Fig. 3B, except that K+ was omitted.

complex of reduced microsomal cytochrome P-450 is shifted to 430 nm (8). The effect of the absence of camphor and K+ on the position of the absorption maximum of the ethyl isocyanide complex of reduced cytochrome P-450 is shown in Fig. 3B and C. In each of these experiments, the absorption spectrum of the carbon monoxide complex of reduced cytochrome P-450 was recorded after bubbling carbon monoxide into the sample cuvette containing ethyl isocyanide. The in- crease in absorbance at 434 nm is seen to correlate qualitatively with a decrease in absorbance at 453 nm and also with a decrease in absorbance at 447 nm in the difference spectrum recorded for the carbon monoxide complex of reduced cytochrome P-450. Thus, in the absence of K+ and camphor, at a constant pH, the absorption maximum of the ethyl isocyanide complex of reduced cytochrome P-450 shifts from 453 to 434 nm, consistent with the conversion of cytochrome P-450 to cytochrome P-420. It should be noted that bubbling the solution with carbon monoxide after forming the ethyl isocyanide complex did not result in

an absorption maximum near 421 nm, in agreement with the experimental results obtained by absolute absorption spectropho- tometry.

The absorption spectrum of the ethyl isocyanide complex of reduced cytochrome P-450 was examined as a function of the pH of the reaction mixture. At pH 7.0 and 8.0, in the presence of camphor and K+, the spectrum of the ethyl isocyanide complex of reduced cytochrome P-450 was essentially the same as in Fig. 3A. However, in the absence of both camphor and Kf the relative intensity of the absorption maxima at, 434 and 453 nm varies with pH and, at a given pH, appears to be somewhat variable de- pending on the amount of dithionite added. It is concluded that, in the absence of both camphor and K+, the intensity of the 434 nm absorption maximum reflects the suseepti- bility of cytochrome P-450 to conversion to cytochrome P-420 under unfavorable conditions, i.e., extremes of pH and excess amounts of reductant.

K, of reduced cytochrome P-450 for ethyl isocyanide. Titration of reduced cytochrome


0.5

0.4 w 0 z a 0.3 m LT g 0.2 m a

0.1

005

A lm?lal oddltion 8 F~nol c,dd,,,on 0.04

0.03

0.02

0.0 I

400 450 500 550 600 650

WAVELENGTH (nmf

FIG. 4. Titration of reduced cptochrome P-450 with ethyl isocyanide in the presence of camphor. The reference cuvette contained 20 mM Tris-chloride buffer, pH 7.4, and 0.1 M KCl. The buffer solution in the sample cuvette contained 2.5 pM cytochrome P-450 and 41 PM camphor. After dithionite reduction of cytochrome P-450 (-*-), the titration was carried out by adding ethyl isocyanide to both cuvettes (-). In the visible region of the spectrum only the initial (A) and final (B) additions of ethyl isocyanide are shown.

FIG. 5. Double-reciprocal plots for the binding of ethyl isocyanide to reduced cytochrome P-450 in the presence of camphor. Cytochrome P-450 concentration, camphor concentration, and K, values were determined as follows: (-a-), 2.5 PM, Xl PM, and 2.2 X W3 M; (-A-), 2.5 PM, 41 pM and 4.1 X 10-a M; (-0-), 2.4 pM, 81 PM,

and 4.9 x 10-Z M. Other experimental conditions are given in the legend to Fig. 4. On adding ethyl isocyanide, the change in absorbance was measured at 453 nm relative to the absorbance of reduced cytochrome P-450 in the absence of ethyl isocyanide.

P-450 with ethyl isocyanide in the presence of camphor results in the changes in the absorption spectrum shown in Fig. 4. The change in absorbance at 453 nm, upon addition of various concentrations of ethyl isocyanide to reduced cytochrome P-450, can be used to calculate the K, of reduced cytochrome P-450 for et,hyl isocyanide. The

FIG. 6. Double-reciprocal plot for the binding of ethyl isocyanide to reduced cytochrome P-450 in the absence of camphor. The buffer employed was 20 mM Tris-chloride, pH 7.4, 0.1 M KCl, and the cytochrome P-450 concentration was 2.2~~. An apparent K, value of 9.0 X 10e6 M was determined. The change in absorbance was measured at 453 nm as stated in the legend to Fig. 5.

apparent K, values, determined from the double-reciprocal plots in Figs. 5 and 6, of 9.0 X lo+ M in the absence of camphor, and approximately 10e3 M in the presence of camphor indicate that camphor is a very good competitor of ethyl isocyanide for a binding site on reduced cytochrome P-450. The K. value in the absence of camphor is comparable to values of l-S.5 X lo+ M reported for microsomal cytochrome P-450 from rabbit liver (5, 8, 10). The rate of formation of the ethyl kocyanide complex with reduced cytochrome P-450 is affected


by the presence of camphor in the reaction mixture, as shown in Table I. At a given ethyl isocyanide concentration, the initial rate of formation of the ethyl isocyanide complex and the final change in absorbance are decreased by increasing the camphor concentration.

Ethyl isocyanide binding by oxidized cytochrome P-450. Ethyl isocyanide coordinates tightly with transition metal ions because it is a very good U-electron acceptor, and it will form complexes with both ferric and

ferrous ions (20). The addition of ethyl isocyanide to oxidized cytochrome P-450 in the presence of camphor results in a change in the absorption spectrum of cytochrome P-450, shown in Fig. 7, indicative of a re- placement of the weak field ligand of the heme iron with a strong field ligand, assumed to be ethyl isocyanide. As a result of this ligand interchange, the spin state of the heme iron is shifted from high to low spin (21). The change in absorbance at 430 nm (EM = 114 X lo3 31-l cm-‘) upon addition

TABLE I

COMPARISON OF THE RATES OF FORMATION OF THE ETHYL ISOCYANIDE COMPLEX OF REDUCED

CYTOCHROME P-450 IN THE ABSENCE AND PRESENCE OF C.IMPHOR~

Sample P-45 (paa) Camphor CUM) EtNC (mn) Initial rate Final (AA/set x ‘103) (AA X 102)

1 1.0 0 2.1 >50 4.2*

2 1.0 1.3 2.1 1.6 5.3 3 1.0 5.0 2.1 1.2 4.7

4 1.0 15.0 2.1 1.0 3.3 1 1.0 0 2.1 >50 4.2

5 1.0 0 0.11 8.9 4.0 6 1.0 0 0.0021 0.21 3.3

a The cuvette contained cytochrome P-450 in 0.1 M Tris-chloride buffer, pH 7.4,0.1 M KCl, and sodium dithionite. The change in absorbance at 453 nm relative to 467 nm on adding ethyl isocyanide to the

cuvette was monitored with time by an Aminco-Chance dual-wavelength/split-beam spectrophotometer in the dual wavelength mode.

* This value may be too small as a result of some cytochrome P-420 formation.

-- Oxldlzed - Oxldlzed

005 + EtNC

A lnltml addition B F~nol addhon

0.04

I I I 400 450 500 550 600 650

WAVELENGTH (nrn)

FIG. 7. Titration of oxidized cytochrome P-450 with ethyl isocyanide in the presence of camphor. The reference cuvette contained 0.1 M Tris-chloride buffer, pH 7.4, 0.1 M KCl. The buffer solution in the sample cuvette contained 3.1 PM cytochrome P-450 and

80 PM camphor (-.-). Ethyl isocyanide was added to both cuvettes during the titration (--). In the visible region, only the initial (A) and final (B) additions of ethyl isocyanide are shown.


of ethyl isocyanide to oxidized cytochrome P-450 can be used to calculate the K, of oxidized cytochrome P-450 for ethyl isocyanide. The double-reciprocal plots shown in Figs. S and 9 indicate that K, is about an order of magnitude larger in the presence of camphor than in the absence of camphor, the K, values being 2.4 X 1OW M and 1.7 X 10e3 AS, respectively. Thus, camphor appears to interfere with the binding of ethyl isocyanide to oxidized cytochrome P-450, as well as reduced cytochrome P-450. K, values reported for the binding of ethyl isocyanide to oxidized microsomal (rabbit liver) cytochrome P-450 are 4-9 X 1O-4 M (6).

EPR spectra of cytochrome P-450. Bacterial cytochrome P-450 in the absence of camphor exhibits a low spin EPR signal with values of g = 2.45, 2.26, and 1.91 (16, 17). Prior work has shown that in the presence of the substrate, camphor, most of the cytochrome is converted to a high spin state, as evidenced by (1) the shift of the Soret absorption maximum from 418 to 392 nm, (2) the large decrease in intensity of the EPR signal centered about g = 2.26, and (3) magnetic susceptibility measurements of the number of unpaired spins in camphor-bound cytochrome P-450 (17). Figure 10 shows the change in the EPR spectrum of low spin oxidized cytochrome P-450 on t,he addition

FIG. 8. Double-reciprocal plot for the binding of ethyl isocyanide to oxidized cytochrome P-450 in the presence of camphor. The experimental

conditions are given in the legend to Fig. 7. The K, value was determined to be 2.4 X lVa M. On

adding ethyl isocyanide, the change in absorbance was measured at 430 nm relative to the absorbance of oxidized cytochrome P-459 in the absence of ethyl isocyanide.

-8 -4 0 4 * I2 16 20 24

I o-2 sic’ lx’

FIG. 9. I)ouble-reciprocal plot for the binding

of ethyl isocyanide to oxidized cytochrome P-450 in the absence of camphor. The sample and refer

ence cuvettes contained a 3.0 PM solution of cytochrome P-450 in 0.1 M Tris-chloride buffer, pH 7.4, and 0.1 M KCI. During the titration, ethyl iso-

cyanide was added to the sample cuvette and the absorption spectrum was recorded. The change in

absorbance plotted is the change in absorbance at 435 nm less that at 415 nm of oxidized cytochrome

P-450 measured in the presence of ethyl isocyanide

relative to the absence of ethyl isocyanide.

g= 246 227 I92

I “I’

I

I

P-450

h

P-450+ E,NC <:,1;1:

9= 2.49 232 IS9

FIG. 10. Electron paramagnetic resonance spec-

tra of oxidized cytochrome P-450 in the absence

and presence of ethyl isocyanide. The cytochrome

P-459 concentration was 70 pM in 2.9 mM Tris- chloride buffer, pH 7.4. Ethyl isocyanide concen-

tration was 0.27 M. Temperature, -170”. Instru-

mental conditions: power, 50 mW; modulation

amplitude, 12.5 G; receiver gain, 2 X 103; scanning

rate, 500 G/min; and, time constant, 0.3 sec.

of ethyl isocyanide. The low spin EPR signal, characteristic of oxidized cytochrome P-450, remains but the lines are both shifted and broadened.


DISCUSSION

The results of t.his study show t,hat, t,he complex formed between ethyl isocyanide and nat)ive bacterial cytochrome P-450 in its reduced state has a single Soret absorption maximum at 4,?3 nm. The absorption spectrum of this complex does not depend on the initial spin state of the oxidized hemoprotein. For example, when titrations of reduced cytochrome P-450 with ethyl isocyanide were carried out, at pH 7.4, and m the presence of Ii+, the absorption spectra were found to be the same whether the oxidized form was initiall? high spin (camphor present) or low spm (camphor absent). As we not,ed in Results, the absence of camphor at pH 5.95 results in the formation of a spectral shoulder near 434 nm in the absorption spectrum of the ethyl isocyanide complex of reduced cytochrome P-450, but other experimental results presented demonstrate that cytochrome P-420 is responsible for this shoulder.

The finding that camphor increases the K, of ethyl isocyanide binding to both oxidized and reduced bacterial cytochrome P-450 indicates that the site of camphor binding t’o cytochrome P-450 is probably near the heme iron. A similar analysis of t,he competition between camphor and mety- rapone for oxidized cytochrome P-450 also indicnt,es that the camphor is bound close t,o the iron atom of the heme ring (22). Even though the absolute spectra and circular dichroism spectra of reduced and the carbon monoxide complex of the reduced form of cytochrome P-450 in the absence and presence of camphor are very similar (17), our results would suggest that, camphor is bound in the vicinity of the porphyrin ring. The conclusion that camphor is close to the heme ring is quite satisfying because the oxygenated intermediate which is formed prior t,o hydroxylation (12) would probably react with t,he first molecule with which it, came into contact, as has been sug- gested by Ullrich and Staudinger (23). If camphor was distant from the sife of oxygen activation, turnover of the enzyme during hydroxylation should result’ in an appreciable amount of “suicide” (24) of the enzyme and this is not. observed.

In t,he following discussion of the ethyl ixocyanide complexes of mammalian and I-‘. putida cytochrome P-450, it should be re- membered that although the protein com- ponents and, therefore, substrate specificit’y of these pigments are different’, their overall spectral properties are similar (17, 25-27). The difference absorption spectrum of the ethyl isocyanide complex of reduced microsomal cytochrome P-450 has absorption maxima in the Soret region at 430 and 455 nnl (4, 5) ; furthermore, the relative int,ensitv of these absorption maxima is sensitive t,o both the pH and t)he ionic strength of the buffer medium (8, 9). It has been found that pre- treatment of animals wit,h phenobarbital or 3-methylcholanthrene, both of which are known to induce hepatic cytochrome l’-450, also affect,s the relative intensity of t,he 455 and 430 nm absorption maxima char- act,eristic of the ethyl isocyanide complex of reduced microsomal cytochrome P-450 (2830). Mannering et aE. (29) showed t,hat,, at a given pH, the intensity ratio of the 45.5 to 430 nm absorption maxima was larger for t,he cytjochrome P-450 associated with liver microsomes from 3-methylcholanthrene-treated animals than for phenobarbital-treated or control animals. On the basis of this and ot,her differences bet,ween 3-methylcholant’hrene and phenobarbital induction of hepatic microsomal cytochrome P-450, it was proposed t.hat a new cytochrome P-450 was formed on induction of liver microsomal cyt,ochrome P-450 with 3-metjhylcholanthrene. Since evidence sug- gested that the new hemoprotein contained a high proportion of the high spin form, it was thought that, the 455 nm absorption maximum could be associated predominantly wit,h the high spin form of cytochrome P-450.

However, Imai and Sato (31) have proposed that the 430 nm maximum in the absorption spectrum of the ethyl isocyanide- reduced microsomal cytochrome P-450 complex is relabed to the effectiveness of et’hyl isocyanide in destroying hydrophobic int’eractions. Various reagents, including organic solvent,s and detergents, convert microsomal cytochrome P-450 to cytochrome P-420 (19), which in its reduced state forms a complex with ethyl isocyanide, having an absorption maximum at 433 nm (4). Thus,

22s GRIFFIN AND PETERSON

with native microsomal cytochrome P-450, the species giving rise to the 430 nm absorption maximum has been attributed to an altered form of cytochrome P-450, which is not cytochrome P-420, but whose environment closely resembles that of cytochrome P-420 (31). The apparent interconvertibility in microsomal cytochrome P-450 of the 430 and 455 nm absorption maxima with changes in pH suggests that the species associated with the 430 nm absorption maximum of the ethyl isocyanide complex can be reversibly formed (8).

The finding that the ethyl isocyanide complex of reduced bacterial cytochrome P-450 exhibits only one Soret absorption maximum at 453 nm supports Imai and Sato’s conclusion that the 455 nm absorption maximum is characteristic of microsomal cytochrome P-450 in its native state (31). It is tempting to speculate that the 430 nm absorption maximum characteristic of microsomal cytochrome P-450 is related to properties of the microsomal membrane with which the cytochrome is so closely associated. One function of the membrane may be that of maintaining the hydrophobic environment of the heme of microsomal cytochrome P-450. If this is so, then alterations in the membrane, caused by various reagents, including such ligands as ethyl isocyanide, may give rise to a species of microsomal cytochrome P-450 in which the heme is in a hydrophilic environment. The ethyl isocyanide complex of this “converted” microsomal cytochrome P-450 is assumed to be responsible for the 430 nm absorption maximum observed with reduced microsomal cytochrome P-450. The bacterial cytochrome P-450 apparently does not depend on membrane to maintain this hydrophobic environment, which may explain why it can be purified and why the ethyl isocyanide complex of its reduced form does not normally display an absorption maximum near 430 nm.

ACKNOWLEDGMENTS

We thank Mrs. M. A. Bailey for her expert technical assistance and Drs. R. W. Estabrook and Y. Ishimura for their advice and encourage-

ment during the course of this work.

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

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