Absorption spectral studies on heme ligand interactions of P-450nor

BIO(?HIMI('A ET BIOPHYSICA A('TA

ELSEVIER

Biochimica et Biophysica Acta 1337 (1997) 66-74

Absorption spectral studies on heme ligand interactions of P-450no r Y o s h i o I m a i a,., N o r i a k i O k a m o t o

~, K a z u h i k o N a k a h a r a

b, H i r o f u m i S h o u n b

Department of Veterinao, Science, Osaka Prefecture Universio', Sakai, Osaka 593, Japan b Institute of Applied Biochemisto', University of Tsukuba, Tsukuba, lbaraki 305, Japan

Received 19 August 1996; accepted 2 September 1996

Abstract Heme-external ligand interactions of P-450.o r were examined spectrophotometrically and compared with those of other P-450s. Most nitrogenous ligands induced type II spectral changes on binding to ferric P-450 .... as did other P-450s. In contrast with other P-450s, 2-methyipyridine and l-butanol induced type I changes in the spectrum of P-450.o r. No spectral interaction of ferrous P-450no r with these ligands was observed. The absorption spectra of the alkyl isocyanide complexes of ferrous P-450 .... exhibited the Soret peak at 427 nm with a slight shoulder at around 455 nm at neutral pH, and this shoulder was intensified as the pH was increased, suggesting that the isocyanide complexes of P-450,o r existed in two states (the 430 and 455 nm states) which were in pH-dependent equilibrium in a similar manner to microsomal P-450s. However, the equilibrium was shifted mostly to the 430 nm state in the complexes of P-450.o~. The findings suggest that P-450no r, especially its ferrous form, has some distinct features from P-450ca m and microsomal P-450s in the distal heme environment. Keywords: Cytochrome P-450; P-450.or; Absorption spectrum; Heme ligand interaction; Alkyl isocyanide-P-450 complex

I. Introduction The P-450s are a superfamily of heme-thiolate proteins involved in the oxidative metabolism of various endogenous and exogenous hydrophobic compounds. They usually constitute electron transfer systems together with component(s) from NAD(P)H and catalyze monooxygenase reactions, where one atom of molecular oxygen is incorporated into or-

" Corresponding author. Fax: +81 722 520341; E-mail: [email protected] t The nomenclature of cytochrome P-450 is adapted from Nebert et al. [1]. The abbreviations used are: P-450nor [2], the product of the CYP55 gene; and P-450¢~m [1], the product of the CYPI01 gene.

ganic substances. The three-dimensional structure has been determined for four bacterial P-450s [3-6], and it has been proposed that general features of the structure are essentially conserved in all P-450 proteins of this superfamily, but many specific sites vary from P-450 to P-450 [4-6]. P-450,o r ( P - 4 5 0 55 in the systematic nomenclature system [1]), purified from the denitrifying fungus Fusarium oxysporum [2], belongs to the P-450 superfamily [7] and has spectral properties characteristic of P-450 [2,8,9]. However, this P-450 has no monooxygenase activity, but catalyzes the nitric oxide reductase reaction, the reduction of NO to N20, by directly accepting electrons from N A D H [2]. Based on spectroscopic and kinetic studies on the reaction of P-450 .... with NO and NADH, a mechanism for the P-450,o r reaction has been proposed [8]. In this

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Y. Imai et al. / Biochimica et Biophysica Acta 133 7 (1997) 66- 74

mechanism, ferric P-450no r (Fe 3+) binds NO to form the Fe3+NO complex, which accepts two electrons from NADH to yield the [Fe3+NO] 2- complex. This complex then reacts with another NO, resulting in the N20 release and the ligand-free Fe 3÷ reproduction. Thus, neither the Fe 2+ NO complex nor the ligand-free Fe z+ state is involved in the catalytic cycle of the P-450no r reaction. On the other hand, the binding of molecular oxygen to ligand-free Fe 2÷ of the substrate-bound form, which yields the Fe2-O2 complex, is the essential step of the monooxygenase reaction catalyzed by the usual P-450s. In addition, the usual P-450 has the binding sites of the lipophilic organic substrate, one of which is located in the structure forming the distal heme pocket [2,10], but P-450,o r does not use an organic compound as a substrate. These suggest possible differences in the structure and environment of the distal heme pocket between P-450,o~ and the usual P-450, although P-450no~ interacts with a small sized ligand, CO, in a similar manner to the camphor-bound form of P450c~~ [9]. Exchange reactions of native ligand for external ones with heme are often utilized to inspect the heme environments of hemoproteins. The absorption spectrum of ferric P-450 characterizes the bonding atom of the sixth ligand to the heme iron and reflects the heme environment [11-13]. In the ferrous state, hepatic microsomal P-450s, when combined with alkyl isocyanide, exists in two interconvertible states (the 455 and 430 nm states), which are in pH-dependent equilibrium [14-16], anti the relative amounts of the two states under a specific condition depend on the P-450 species [ 15-17], probably reflecting the differences in the heme environments of each P-450. On the other hand, the ethyl isocyanide complex of ferrous P-450ca m exists only in the 455 nm state [18]. In this study, we examined the spectral interactions of various external ligands with P-450°o ~ and compared them with those of the usual P-450s. Most nitrogenous ligands, including imidazoles, could bind P450,o r in a manner similar to the case with the usual P-450s in the ferric state but did not interact with ferrous P - 4 5 0 n o r. The alkyl isocyanide complexes of ferrous P-450,o~ existed in the two states, as is the case with other P-450s, but the equilibrium was shifted toward the 430 nm state to a large extent which has never been observed for other P-450s.

67

These findings suggest that the heme environment of P - 4 5 0 n o r has features that are generally similar to, but some that are distinct, from those of other P-450s.

2. Materials and methods P-450 .... was isolated from F. oxysporum and purified according to the published procedures [2]. P-450~a,n was prepared from transformed Escherichia coli cells, as previously described [19]. P-450 2C2, P-450 2C14, and P-450 2El were prepared from transformed yeast cell according to the published procedures [20-22]. P-450 1A2 and P-450 2B4 were purified from rabbit liver microsomes as previously described [23]. t- and n-Butyl isocyanides were purchased from Aldrich Chemical Co, Inc. (Milwaukee, WI, USA). Ethyl isocyanide was synthesized by the method of Jackson and McKusick [24]. Other chemicals used were from the same sources as previously described or of the highest qualities commercially available [ 16]. Spectrophotometric measurements were carried out with a Jasco Ubest 50 spectrophotometer at room temperature.

3. Results

3.1. Interaction of nitrogenous ligands with

P-450no r

Ferric P - 4 5 0 n o r has a low- and high-spin mixedtype spectrum of P-450 [2]. When 1-methylimidazole was bound to ferric P-450no r, an spectrum characteristic of a nitrogenous-ligand-bound form of ferric P-450 was observed (Fig. 1): the Soret band (Table 1) was red-shifted, as compared with that of a native low-spin form of P-450 (Areax, 415-418 nm) [I 1-16] and the /3 band (/~max' 544 nm) was more intense than the a band. The dissociation constant (K~) of 1-methylimidazole was determined from the spectral titration of P-450no r with I-methylimidazole, and the K~ value (Table 1) was comparable to those reported for microsomal P-450s [11,13,25] and P - 4 5 0 c a m (Hada, T. and Imai, Y., unpublished data). Similar spectral changes (type 1I change) were observed on binding of 2-methylimidazole, 4-methylimidazole, 4phenylimidazole, pyridine, 3-methylpyridine, 4-meth-

68

Y. lmai et al. / Biochimica et Biophysica Acta 1337 1997) 66-74

Ji

0.5 ~ (D

cO0.4 t~ c~ ~0.3.Q

< 0.2---. 0.1-

0.5. -

400

.L-

500 Wavelength

,

600 (nm)

700

~0.4r'~

Fig. 1. Absorption spectra of l-methylimidazole complex of P-450nor. P-450,o ~ was dissolved in 100 mM potassium phosphate buffer (pH 7.25). The spectra were measured in the presence of 50 mM 1-methylimidazole. Solid line, oxidized form; dashed line, dithionite-reduced form.

ylpyridine, and 1-octylamine to ferric P-450 .... (Table 1). These nitrogenous ligands, except for 2methylimidazole, had K s values on the same order o f magnitude as I - m e t h y l i m i d a z o l e (Table I). On the other hand, when 2-methylpyridine was added to P-450no r, a type I spectral change was induced (Fig. 2), in contrast with the spectral changes observed in

Table 1 Spectral characteristics of external ligand complexes of ferric P-450.o r Sixth ligand Soret band Apparent dissociation A.... (nm) constant ~ (mM) Nitrogenous

1-Methylimidazole 2-Methylimidazole 4-Methylimidazole 4-Phenylimidazole Pyridine 2-Methylpyridine 3-Methylpyridine 4-Methylpyridine 1-Octylamine

424 423 424 423 421 394 421 422 424

l 100 7 1 7 = 100 3 3 n.e.

430

0.1

lsocyanide

n-Butyl isocyanide

I.

-,

/

\

0.2 ~:"

x4 e

400

560 Wavelength

600 (nm)

700

Fig. 2. Titration of ferric P-450.,,r with 2-methyipyridine. P450nor was dissolved in 100 mM potassium phosphate buffer (pH 7.25). The final concentrations of 2-methylpyridine were 0 (a), 20 (b), 60 (c), 120 (d), and 200 (el mM. Absorption differences between (b)-(e) and (a) are shown in (B)-(E).

microsomai P-450s and P-450ca m [11,13,26]. NO spectral change was observed on addition of 2-phenylimidazole (100 mM), which is k n o w n to induce an abnormal change in the spectrum o f P-450c~ m and microsomal P-450s [ 12,13]. When the l - m e t h y l i m i d a z o l e c o m p l e x of ferric P-450no r was reduced with dithionite, the spectrum was changed to that of the external ligand-free form of ferrous P-450,o r (Fig. 1), indicating that ferrous P-450no r did not bind l - m e t h y l i m i d a z o l e at a concentration of 100 mM. This finding was observed for the other nitrogenous ligands examined. It was confirmed from the spectrum of the C O c o m p l e x that no conversion to a denatured P-420 form occurred on the reduction of the nitrogenous-ligand-bound form of ferric P-450no r (data not shown). 3.2. I n t e r a c t i o n o f o x y g e n o u s ligands with P - 4 5 0 ....

Oxygenous

l-Butanol 1-Propanol

394 394

= 100 = 300

Concentration of the ligand which induced a half-maximal change in the spectrum independently of the mechanism of the spectral change. Not estimated.

It has been reported that, in the ferric state, the spectra of 1-propanol and 1-butanol c o m p l e x e s of P-450 are essentially identical with that of native low-spin P-450 [ 1 1 - 1 3 ] and, thus, the addition of these alcohols to the high- or mixed-spin form of P-450 induces a reverse type I spectral change. H o w -

69

Y. Imai et al. / Biochimica et Biophysica A eta 133 7 (1997) 6 6 - 74

ever, when these alcohols were added to ferric P450~o~, the spectrum changed in that the high-spin component was increased (type I change) (Fig. 2). Type I spectral change was also observed on adding ethanol to ferric P-450:,o~ (data not shown). These alcohols did not affect the spectrum of P-450,,,~ in the ferrous state.

,

a

Jl b

0.8I

'd 0.6f-

3.3. Interaction of alkyl isocyanides with P-450,,,,

t~

o

When n-butyl isocyanide was bound to ferric P450,,,r, an absorption spectrum having peaks at 551, 430, and 369 nm was observed (Table 1). A single broad band in the visible region and a red-shifted Soret band as compared with the nitrogenous-ligandbound form are characteristic of the spectrum of the alkyl isocyanide complex of ferric P-450 [13,15,18]. The K~ value estimated from the spectral titration (Table 1) was comparable to those reported for the other P-450s [13,18,27]. On addition of t-butyl or ethyl isocyanide to ferric P-450 ..... similar spectral changes were observed, although these isocyanides had much lower affinities than n-butyl isocyanide. In the ferrous state, the n-butyl isocyanide complex of ferrous P-450 .... had a single Soret peak at 427 nm at pH 6.5, and the o~ band (A,,~x, 558 nm) was more intense than the /3 band (A,,~, 528 nm) (Fig. 3). No band was detectable at around 455 nm, which was the wavelength where the ethyl isocyanide complex of other P-450s had the absorption maximum [ 15,17,18,28]. However, when the pH was increased to higher than 7.5, a :dight shoulder appeared at around 455 nm (Fig. 3). This shoulder of the absolute spectrum could be visualized as a peak by recording the spectral difference between pH 8.0 and pH 6.5 (Amax. 455 nm) or that between the n-butyl isocyanide complex and the free form at pH 8.0 (Am,x, 461 nm) (data not shown). Concomitant pH-dependent alterations could also be observed in the visible region. The t-butyl and ethyl isocyanide complexes of ferrous P-450no r exhibited similar absorption spectra to that of the n-butyl isocyanide complex, but the shoulder at around 455 nm was a little more obvious in the t-butyl and ethyl isocyanide complexes (Table 2). These findings suggest that the alkyl isocyanide complexes of ferrous P-450~o ~ exist in two interconvertible states (the 430 and 455 nm states), as do those of microsomal P-450s, but the equilibrium

t

0.4-

'

x4 '

a

,

i 0.2-/ 2

i

..

'~ ~~/.--.~,, ~_~

400

..... s O o

Wavelength

.....

600

(nm)

Fig. 3. Absorption spectra of n-butyl isocyanide complex of ferrous P-450 ..... at different pH values. P-450 .... was dissolved in 200 mM potassium phosphate buffer. After addition of 0.5 mM n-butyl isocyanide, the P-450 was reduced with dithionite and the spectrum was recorded, a (solid line), pH 6.5: b (dashed line), pH 7.0: c (solid line), pH 7.5, d (dashed line), pH 8.0.

between the two states shifts mostly toward the 430 nm state, even at pH 8.0. The spectral changes of ferrous P-450,o r induced by the alkyl isocyanides were examined quantitatively and the titration curves thus obtained (Fig. 4) indicated that the alkyl isocyanides interacted with ferrous P-450 .... in a simple and normal manner, as did P-450¢,,,, and microsomal P-450s [15,18,25]. The K~ values of the alkyl isocyanides for ferrous P-450°, r were always larger than those for other P-450s, when compared for the same alkyl isocyanide (Table 2). The K~ value of n-butyl isocyanide for ferrous P-450,o r was smaller by one order of the magnitude than those of t-butyl and ethyl isocyanides (Table 2).

3.4. Binding of n- and t-butyl isocyanides with the .ferrous forms c?["P-450 .... and microsomal P-450s In contrast with the alkyl isocyanide complexes of P-450 .... , the ethyl isocyanide complex of ferrous P-450 .... exhibits a single Soret absorption peak at 453 nm and the spectrum is unaffected by changes in

70

Y. lmai et al. / Biochimica et Biophysica Acta 1337 (1997) 66-74

lO0

,.1"

80=

20 0

....

1.2~

. . . . . .

._.~,

..........

_.3

kog[Alkyl isocyanide] (M)

Fig. 4. Titration of ferrous P-450s with n- or t-butyl isocyanide. P-450nor and P-450cam were dissolved in 100 mM potassium phosphate buffer (pH 7.25). P-450 1A2 was dissolved in 200 mM potassium phosphate buffer (pH 7.25) containing 20% glycerol. AAmax was obtained from double reciprocal plots of the absorbance change (AA) at 426 nm (P-450,or), 429 nm (P-450 1A2), or 453 nm (P-450cam) induced by the addition of n-butyl isocyanide against its concentrations. (O) P-450,o,: (11) P450 .... ; (A) P-450 IA2. Titration of P-450 .... with t-butyl isocyanide is shown as (©). For reference, titration of P-450 1A2 with ethyl isocyanide ( × ) is taken from Ref. [23].

pH between 6 and 8 [18]. Thus, the s p e c t r u m was e x a m i n e d when P - 4 5 0 ..... was c o m b i m e d with butyl isocyanides. Both n- and t-butyl isocyanide c o m plexes s h o w e d spectra quite similar to that o f the ethyl isocyanide c o m p l e x , h a v i n g a single peak with no detectable band at around 430 nm in the Soret region and a m o r e intense /3 band (Am, x. 549 nm) than the o~ band (Am,~x, 575 nm) in the visible region, independent o f pH b e t w e e n 6.5 and 8.0 (Table 2). T h e n- and t-butyl isocyanide c o m p l e x e s o f microsomal P - 4 5 0 s , such as P - 4 5 0 s I A 2 , 2B4, 2C2, 2 C ! 4 , and 2 E l , exhibited two Soret peaks at a r o u n d 430 and 455 nm, the relative intensities o f w h i c h depended on the species o f P - 4 5 0 and were affected by c h a n g e s in pH (Table 2) in a similar m a n n e r to the case reported for their ethyl isocyanide c o m p l e x e s [24,28]. W h e n c o m p a r i n g the n- and t-butyl isoc y a n i d e c o m p l e x e s o f each P-450, the f o r m e r had lower relative intensities o f the 455 n m peak to the 430 n m peak than the latter at e v e r y pH e x a m i n e d

Table 2 Spectral characteristics of alkyl isocyanide complexes of ferrous P-450 cytochromes P-450 P-450 ....

P-450c~m

I A2

2B4

2C2

2C 14

2El

Alkyl n-Butyl t-Butyl Ethyl n-Butyl t-Butyl Ethyl n-Butyl t-Butyl Ethyl n-Butyl t-Butyl Ethyl n-Butyl t-Butyl Ethyl n-Butyl t-Butyl Ethyl n-Butyl t-Butyl Ethyl

K

Soret absorption band Amax(nm)

pH dependence (Ratio ")

(mM)

427 426 426

+ + + _ _ _ + + + + + + + + + + + + + + +

0.03 0.4 0.3 0.01 0.06 0.009 0.005 0.02 0.02 0.003 0.(X)3 0.004 0.008 0.02 0.002 n.d. " n.d. n.d. 0.002 0.005 0.(X)3

429, 429, 430, 430, 430, 430, 429, 429, 430, 429, 430, 429, 430, 430, 429,

453 452 453 452 452 456 456 457 456 456 457 455 456 456 454 457 457 457

" Ama~(455 nm peak)/Am,x(430 nm peak) at pH 7.25. b No band was detectable at around 430 nm. c Not determined.

( < 0.1 ) (0.12) (0. I 1) (_ h) (_ h) (_ b) (0.60) (0.69) (0.96) (0.46) (0.82) (0.41) (0.30) (0.58) (0.53) (0.67) (0.87) (0.58) (0.27) (0.34) (0.35)

References This paper This paper This paper This paper This paper [I 8] This paper This paper [25] This paper This paper [15], this paper This paper [ 16], this paper [28] This paper This paper This paper This paper [ 16], this paper This paper

Y. Imai et al. / Biochimica et Biophysica Acta 1337 (1997) 6 6 - 7 4

(6.5 to 8.0) (Table 2). However, both butyl isocyanide complexes showed lower relative intensities than the ethyl isocyanide complex in the case with P-450 1A2, while the ethyl isocyanide complex had the lowest, with P-450 2B4. From the spectral titration, simple and normal interactions of butyl isocyanides with P - 4 5 0 ~ m and microsomal P-450s were demonstrated (Fig. 4). Affinities of n-butyl isocyanide for microsomal P-450s and P-450ca m were not lower than those of t-butyl isocyanide (Table 2).

3.5. Competition of n-bu~.'l isocyanide and CO for P-450~,, To confirm the reversible binding of n-butyl isocyanide to the ferrous heine of P-450no ~, the competition of the isocyanide with CO was examined. When CO was thoroughly bubbled through to the n-butyl isocyanide complex of ferrous P-450no ~ formed by

o.8 ?. . . .

0.6

¸

A

B -

.

C

-I

i i

(D E

~o.40 o3 <~

'

"/

"

.[ .

0

.--:-.'-: - ' ~ : ' J 400 450 5004'~)

4.50

Wavelength

0.8" ? ,J

0.6 ort~ .o ~

..113

0.4"

0.2.

o ,iOo

500 Wavelength

600 (nm)

Fig. 6. Conversion of n-butyl isocyanide complex of P-450.o r to denatured form. P-450 ..... was dissolved in 100 mM potassium phosphate buffer (pH 7.25). After addition of 0.5 mM n-butyl isocyanide, the P-450 was reduced with dithionite and the spectrum was recorded (dashed line). Then, phenol was added (final concentration, 0.8%) and the spectrum of the solution was recorded alter allowing it to stand for 5 min (solid line).

-

i

0.2 . . . .

71

|//

i I ~

400

\, '\ - 450

500

(rim)

Fig. 5. Competition of n-butyl isocyanide and CO for P-450,0 r. P-450nor was dissolved in 100 mM potassium phosphate buffer (pH 7.25). After the first addition of n-butyl isocyanide or CO, the P-450 was reduced with dithionite and the spectrum was recorded. Then, the second addition of CO or n-butyl isocyanide was carried out and the spectrum was recorded. Panel A: first addition, 0.3 mM n-butyl isocyanide (dashed line); second addition, bubbling of CO through the sample (solid line). Panel B: first addition, bubbling of CO through the sample (solid line); second addition, 0.4 mM n-butyl isocyanide (dashed line). Panel C: first addition, 50 /zl of CO saturated solution in water which was contained in 500 #1 of the phosphate buffer instead of the addition (dotted line); second addition, 0.2 mM n-butyl isocyanide (chained line), and then, bubbling of CO through this sample (solid line).

the addition of the isocyanide at 0.3 mM, P-450no r was mostly converted to the CO complex (Fig. 5A). Conversely, when the isocyanide was added to the CO complex produced by bubbling CO through it, only a part of the CO complex was converted to the isocyanide complex (Fig. 5B). On the other hand, when the CO complex was produced by adding CO at about 0.1 mM (one-tenth of CO-saturated solution in water) to ferrous P-450nor, about one half of the CO complex was converted to the isocyanide complex on the addition o f n-butyl isocyanide. The CO complex was mostly recovered by bubbling CO through to this solution (Fig. 5C). The relative amounts of the CO and isocyanide complexes thus formed are reasonable from the dissociation constants of CO [9] and n-butyl isocyanide for P-450,o r and the concentrations of both chemicals.

3.6. Coneersion of butrl isocyanide complexes to denatured form It has been reported that the ethyl isocyanide complex of the denatured form of P-450ca m exhibits

72

Y. lmai et al. / Biochimica et Biophysica Acta 1337 f / 9 9 7 ) 6 6 - 7 4

an absorption spectrum having a Sorer peak at 432 nm in the reduced form [18]. The addition of phenol. the denaturant of P-450~,.n [18], at 0.8% to the n-butyl isocyanide complex of ferrous P-450,,,r at pH 7 caused changes in the absorption spectrum: the Sorer peak was shifted from 427 nm to 434 nm, which was accompanied by changes in the visible region (Fig. 6). Similar conversions of the spectrum were observed on the addition of other denaturants of P-450, such as KSCN or guanidine hydrochloride [29], to the t-butyl isocyanide complex of ferrous P-450 .... . These findings confirm that the Sorer absorption band at 427 nm of the butyl isocyanide complexes of ferrous P-450 ..... is not ascribed to its denatured form.

4. Discussion When the ferrous heme of P-450 is combined with an external ligand, an absorption band is usually observed at 440 to 460 nm in the Sorer region [13,15-18]. In addition, some complexes exhibit an additional Sorer band at 420 to 430 nm, as seen in the spectra of the alkyl isocyanide and pyridine complexes of microsomal P-450s [ 13,15-17], indicating the presence of two coordination states in the case of these complexes. On the other hand, P-450~.~,,, exhibited a single Soret peak at 453 nm, when combined with n- or t-butyl isocyanide (Table 2), as well as with ethyl isocyanide [18]. The complex of ferrous P-450ca m with a nitrogenous ligand such as pyridine or metyrapone, also shows a single peak at around 445 nm in the Soret region [30,31]. P-450 ..... is the only P-450 species ever examined for which the 455 nm peak of the alkyl isocyanide complexes is hardly detectable at neutral pH and, moreover, binding of a typical nitrogenous ligand such as the imidazoles to the ferrous form is not observable. Which of the two possible coordination states the complex prefers may be determined by the structure of the distal heme pocket, which is believed to be generally conserved but has divergences among P-450 species [4-6]. It has been suggested that the interconversion between the 455 and 430 nm states of the ethyl isocyanide complex is accompanied by a change in the protein conformation [32,33]. X-ray crystallographic analysis of the structure of

the metyrapone complex of ferric P-450 ....... has been revealed that one of the two pyridine-nitrogen coordinates with the heme iron oriented normal to the heme plane [37]. Comparing this structure with the crystal structure of ferric P-450c~ m and its ferrous CO complex [2,38], it is reasonable to assume that the structure of the metyrapone complex of the ferric P-450 ...... is retained in its ferrous form. We assume that the 455 nm state of the alkyl isocyanide complex has a similar structure to the metyrapone complex of P450 ...... . For the structure of the 430 nm state, the following possibilities may be assumed: (a) For some reason the coordination bond between the carbon atom of the isocyanide and the heme iron is stretched and strained. A similar situation has been discussed for the spectra of P-450 liganded by molecules having a bulky group which may sterically hinder the normal coordination [16,3 I]; (b) the coordination bond is so extremely bent from the heine normal that it does not keep the normal character, such as the 455 nm state of the ethyl isocyanide complex of P-450 ...... [26]: (c) the 5th coordination bond is affected, which includes a possibility of reversible displacement of the thiolate anion, by the 6th coordination and the ionization of a certain residue through protein conformation. In any case, the conformation of the P-450 protein may be related to determining how the alkyl isocyanide sits in the heme pocket in order to coordinate with the heine iron and which of the 430 and 455 nm states the complex prefers. Observations suggesting a certain conformational difference between the two states of the ethyl isocyanide complex have been reported [32,33]. Shifts in the equilibrium between the two states of the ethyl isocyanide complex by changes in the heme environment have also been observed [25,28]. P-450 I A2, which can metabolize polycyclic hydrocarbons, binds various hydrocarbons at the substrate site, and the binding of the hydrocarbons causes a shift in the equilibrium dependent on the size and shape of the hydrocarbons [25]. When the conserved threonine of P-450 2C2 is replaced with other amino acids, the equilibrium between the two states is also shifted [28]. This study (Table 2) confirmed that the equilibrium between the two states of the alkyi isocyanide complexes of microsomal P-450s was affected by the structure of the alkyl group [36]. The effect occurred in a different manner for the tour microsomal P-450s (P-450s

K hnai et al./Biochimica et Biophysica Acta 1337 (1997) 66-74

1A2, 2B4, 2C2, and 2El) and was dependent on the species of P-450s, which might be related to the structure of the substrate sites of each P-450. However, further work is needed to determine the coordination characters and structures of the 430 and 455 nm states of the alkyl isocyanide complex of P-450. In the ferric state, P-450°o r interacted normally with the heme ligands except for 2-methylpyridine and alcohols, which induce a type I spectral change (Table 1). 2-Methylpyridine is known to be a weak nitrogenous ligand, and the complexes of microsomal P-450 and P - 4 5 ( ) ~ m with 2-methylpyridine exhibit an absorption spectrum deviating from that with the typical nitrogenous ligand, such as pyridine [11,26]; the a band is seen as a distinct peak in the case of the 2-methylpyridine complexes of P-450 I A2 and P-450 . . . . while only a shoulder is observed with the pyridine complexes. The present study (Fig. 2) indicated that the 2-methylpyridine-nitrogen was too weak to serve as the sixth ligand of the ferric P-450,,,r heme and, furthermore, due to the hydrophobic nature of this compound, a water molecule was probably excluded from the vicinity of the sixth ligand position. X-ray crystallographic analysis has revealed that the sixth coordination position of ferric P-450~ m is occupied by an oxygen atom from water or hydroxide anion in the low-spin state (the substrate-free form) [34] but is vacanl in the high-spin state (the camphor-bound form), in which camphor is bound to the P-450c~ m protein by a hydrogen bond between the carbonyl oxygen atom of camphor and the sidechain hydroxyl group of Tyr-96. The hydrophobic surface of the camphor molecule is oriented toward the distal surface of the heme [2]. When alcohols are added to the ferric form of microsomal P-450 or P-450~ m containing the high-spin component, the low-spin state is increased, probably due to coordination of the oxygen atom of the alcohols as the sixth ligand [11-13]. This orientation of the alcohol molecule may be possible from binding of its alkyl chain to the substrate binding site of P-450 via hydrophobic interaction. On the other hand, the possibility has been suggested that, when alcohols having a large alkyl chain are added, the water molecule is excluded from the ~icinity of the sixth ligand position of P-450 and, tbus, the high-spin component of P-450 is increased [35]. A similar situation to this may occur in the case with the interaction of alcohols

73

with P-450no r which does not use a hydrophobic molecule as a substrate. In as far as the ligand exchange reactions are compared between P-450,o r and other P-450s, it seems that the distal heme environment of P-450no r has generally similar but some distinct features from those of other P-450s. Studies utilizing site-directed mutageneses at the putative distal region are in progress in our laboratory and the results thus far support this suggestion (Okamoto, N., Imai, Y., and Shoun, T., unpublished data). X-ray crystallographic analysis that is under way [39] should elucidate this problem.

Acknowledgements This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas (07280224) from the Ministry of Education, Science and Culture of Japan to Y.I.

References [1] Nelson, D.R., Kamataki, T., Waxman, D.I., Guengerich, F.P., Estabrook, R.W., Feyereisen, R., Gonzalez, F.J., Coon, M.J., Gunsalus, I.C., Gotoh, O, Okuda. K. and Nebert, D.W. (1993) DNA Cell Biol. 12, 1-51. [2] Nakahara, K., Tanimoto. T., Hatano, K., Usuda, K., and Shoun, H. (1993) J. Biol. Chem. 268, 8350-8355. [3] Poulos, T.L., Finzel, B.C. and Howard, A.J. (1987) J. Mol. Biol. 195, 687-700. [4] Ravichandran, K.G., Boddupalli, S.S., Hasemann, C.A., Peterson, J.A. and Deisenhofer, J. (1993) Science 261, 731736. [5] Hasemann, C.A., Ravichandran, K.G., Peterson, J.A. and Dcisenhofer, J. (1994) J. Mol. Biol. 236, 1169-1185. [6] Cupp-Vickery, J.R. and Poulos, T.L. (1995) Nature Struct. Biol. 2, 144-153. [7] Kizawa, H., Tomura, D., Oda, M., Fukamizu, A., Hoshino, T., Gotoh, O., Yasui, T. and Shoun, H. (1991) J. Biol. Chem. 266, 10632-10637. [8] Shiro, Y., Fujii, M., lizuka, T., Adachi, S., Tsukamoto, K., Nakahara, K. and Shoun, H. (1995) J. Biol. Chem. 270, 1617-1623. [9] Shiro, Y., Kato, M., lizuka, T., Nakahara, K. and Shoun, H. (1994) Biochemistry 33, 8673-8677. [10] Gotoh, O. (1992) J. Biol. Chem. 267, 83-90. [1 I] Yoshida, Y., Imai, Y. and Hashimoto-Yutsudo, C. (1982) J. Biochem. 91, 1651-1659.

74

Y. hnai et a l . / Biochimica et Biophvsica Acta 1337 (1997) 66-74

[12] Dawson, J.H.. Andersson, L.A. and Sono, M. (1982) J. Biol. Chem. 257, 3606-3617. [13] White, R.E. and Coon, M.J. (1982) J. Biol. Chem. 257, 3073-3083. [14] Imai, Y. and Sato, R. (1967) J. Biochem. 62, 464-473. [15] Hashimoto-Yutsudo, C., Imai. Y. and Sato, R. (1980) J. Biochem. 88, 505-516. [16] lmai, Y., Fukuda, T., Komori, M. and Nakamura, M. (1994) Biochim. Biophys. Acta 1207, 49-57. [17] Ryan, D.E. and Levin, E. (1990) Pharm. Ther. 45, 153-239. [18] Griffin, B.W. and Peterson, J.A. (1971) Arch. Biochem. Biophys. 145, 220-229. [19] Imai, M., Shimada, H., Watanabe, Y., Matsushima-Hibiya, Y., Makino, R., Koga, H., Horiuchi, T. and Ishimura, Y. (1989) Proc. Natl. Acad. Sci. USA 86, 7823-7827. [20] Imai, Y. (1988) J. Biochem. 103, 143-148. [21] Imai, Y., Uno, T. and Nakamura, M. (1990) J. Biochem. 108, 522-524. [22] Imai, Y. (1987) J. Biochem. 101. 1129-1139. [23] Imai, Y., Hashimoto-Yutsudo, C., Satake. H., Girardin, A. and Sato, R. (1980) J. Biochem. 88, 489-503. [24] Jackson, H.L. and McKusick, B.C. (1955) in Organic Syntheses (Cairns, T.C., ed.), Vol. 35, pp. 62-64, Wiley, New York. [25] Imai, Y. (1982) J. Biochem. 92, 77-88. [26] Shimizu, T., Iizuka, T., Shimada, H., Ishimura, Y.. Nozawa,

[27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39]

T. and Hatano, M. (1981) Biochim. Biophys. Acta 670. 341-354. Nishibayashi, H., Omura, T. and Sato, R. (1966) Biochim. Biophys. Acta 118, 651-654. Imai. Y. and Nakamura, M. (1989) Biochem. Biophys. Res. Commun. 158, 717-722. Imai, Y. and Sato, R. (1967) Eur. J. Biochem. 1,419-426. Griffin, B.W. and Peterson, J.A. (1972) Biochemistry 11, 4740-4746. Peterson, J.A., Ullrich, V. and Hildebrandt, A.G. (1971) Arch. Biochem. Biophys. 145, 531-542. lmai, Y. and Sato, R. (1968) J. Biochem. 63, 370-379. Imai, Y. and Mason, H.S. (1971) J. Biol. Chem. 246, 5970-5977. Poulos. T.L., Finzel. B.C. and Howard, A.J. (1986) Biochemistry 25, 5314-5322. Yoshida. Y. and Kumaoka, H. (1975) J. Biochem. 78, 455 -468. Ichikawa, Y. and Yamano, T. (1968) Biochim. Biophys. Acta 153, 753-765. Poulos, T.L. and Howard, A.J. (1987) Biochemistry 26, 8165-8174. Raag, R. and Poulos. T.L. (1989) Biochemistry 28, 75867592. Nakahara. K., Shoun. H., Adachi, S., lizuka, T. and Shiro, Y. (1994) J. Mol. Biol. 239. 158-159.