Cytochrome P-450 oxygen intermediates and reactivity at subzero temperatures

Journal

of Molecular

@ ELsevia

Sequoia

CYTOCHROME AT SUBZERO

CLAUDE PASCALE INSERT

KRISTOFFER

BP 5051,

299

7 (1980)

Lausanne

-

Pd50 OXYGEN TEMPERATURES

BONPILS, DEBEY U-12,$

Catalysis.

S-A..

34033

299 - 308 Print& in the Netherlands

INTERMEDIATES

K_ ANDERSSON, Monfpellier

Cedex

AND

PATRICK

REACITVITY

_MAUREL

and

(FMIXC~}

Summary The oxy-ferro compound is the last stable intermediate of the cytochrome P450 reaction cycle leading to substrate hydroxylation. it has been stabilized at subzero temperatures for two systems, the bacterial camphor hydroxylase and the microsomal drug hydroxylase. Their stability, spectra, decomposition and reactivity are compared.

introduction C=C and C-G bonds can be attacked by ‘activated’ species of oxygen produced by certain chemical systems which include a transition metal ion. a complexing agent and molecular oxygen It is thought that the ‘activated oxygen’ thus produced iucIudes entities such as 0;) OH-, 05, OzLT-, HzOz and ‘oxene’, which are free or in a complexed form ]I- 3]_ A great number of similar reactions occur at the iron center of heme containing monooxygenases. These enzymes are collectively called cy@ chrome P-450 and represent a class of analogous proteins, widely distributed in numerous organs of mammals, in plants and in microorganisms [4] _ They differ in structure, molecular weight, sequence, antigen determinants, and specificity, but they all have a common specific structure at the heme pocket where sulfur is Iigand to the iron. This pocket binds both the substrate to be hydroxylated and the mclecular oxygen which is activated through uptake of a second electron by the Fe”/Oz pair. Two electrons are supplied from NAD(P)H via a step by step short specific electron transport chain which is organized as a multiprotein complex [5,6] _ The advantages of such a compact system are obvious: stereospecificity of the reaction and minimal loss of free active spies and radicals able ~KB diffuse into the surrounding medium. Similarities in the sequence of events.occurring at the iron center of the various P-250’s was.postulated many years ago. The generally accepted reastion cycle is given iu Fig. 1 [7, 8]_ The peroxide and NAJBPH/O, pathways

300

R’OOH

Fig.

1. ProPused

reaction

cycle

F-.

+* RI-4

OT cytochmme

P*s_

Fe3+

=

ferric

protein;

RH

=

substrate.

may well coexist in each system and their respective importance be affected by the substrate and environment [9] _ This postulated reaction pathway is an oversimplification as it omits many unstable intermediates - especially after the uptake of the second electron_ In the present work, we have exploited the methods of cryoenzymolo,T [lo] to stabilize the putative intermediates on the cytochrome P-450 reaction pathway. This study is centered on the heme-oxygen compound of two different systems which will be compared, namely, the soluble and specific camphor hydroxylase from Pseudomonas putida [ 111 and the membranebound, non-specific-drug monooxygenase from liver microsomes.

Materials and methods All reactions are performed in water/ethylene glycol (1: 1 v/v) or water/ glycerol (1:l v/v) mi..tures. Water is buffered with phosphate, cacodylate, or Tris buffer and contains suitable concentrations of salts_ The protonic activity (pal!) of the mixed solvent was established at various temperatures

1121.

Optical spectra are recorded on a classical Aminco-Chance DW2 apectrophotometer modified for subzero temperature thermostatisation [ 131. A special cell thermostatable at subzero temperature and coupled with flash photolysis can be mounted in the cell holder compartment of the spectrophotometer [14]_ A stopped-flow device has been similarly designed for rapid mixing at subzero temperature with limited dead-time (5 ms) [15]. Both devices use the optics and electronic detection of the AminceChance spectrophotometer_

301

prepared according to The bacterial cytochrome P-450 (P450 ,,) Gunsalus and Wagner [16] was a generous gift of Prof. I. C. Gunsalus. The liver microsomes of phenobarbitaI treated rats and rabbits are prepared and washed according to Van der Hoeven eL 2L [I71 and stored frozen in concentrated suspension used within one month. The cytochrome P-450 is soiubilized and puri%d from phenobarbital treated rabbits according to the procedure of Kaugen and Coon ]l8]. The preparation m&&ted as a single polypeptide on an SDS-polyacrylamide gel and the specific heme content was 12 - 16nmol per mg protein.

Rsu1t.s and discussion Oxygencted

compound us a functiomd iizfermediute An oxygenated compound of reduced bacterial cytochrome P450 with, or without, camphor was discovered long ago and characterized by various analytical methods [7] . Its functional role in the overall hydroxylation was demonstrated. However, its structure and reactivity could not be fully studied owing to its rapid nonenzymatic autoxidation, especially in the absence of substrate. Owing to its high activation energy of autoxidation (75 kJ/mol) the oxygenated compound can be stabilized for hours and even days in fluid hydro-organic mixtures, even in the absence of substrate 1191. Its further separation from the reagents (dithionite or photochemical system) used for its reduction can be achieved by chromatography at subzero temperature. This leads to a concentrated solution of the pure oxy-compound which can then bc used as a ‘starting’ reagent [ZO] . A similar oxygenated compound was postulated in the reaction cycle of the microsomal cytochrome P-450. It was identified by Estabrook and coworkers during the steady-state turnover of the whole microsomal multienzyme system [21] . Its absolute spectrum on a purified solubilized preparation was obtained by Guengerich et al. [22] using the stopped-flow technique. We have r@cently been able to stabilize this compound by the addition of 0, at -30 “C to a fluid solution of reduced cytochrome in a lrl (v/v) mixture of water and glycerol. A prebrninary report is published elsewhere [ 231. Its absolute spectrum, together with those of the oxidized and reduced forms, is presented in Fig. 2 and its spstral characteristics, together with those of the substrate-free and -bound oxy-ferro compound of P450,_ are given in Table 1. The difference spectrum Fe’* f Oa. minus Fe’+ (Fig. 3) is very similar to the ‘Complex II’ observed by stopped flow by Guengerich et aL [22] at 4 “C in aqueous medium, as well as to the difference spectrum originally observed by Estabrook 1211. We also obtained under the same conditicns of solvent and temperature an identical differential spectrum by addition of Oz to a fully reduced microsomal suspension [24]. In the latter case, however, the oxycompound was not stable and decayed into the ferric cyto(i)

302

360

400

440

480

!i2O

560

600

640

600 hinnm

Fig. 2 _ Absolute spectra of solubilized microsomal cytochrome Pa, _ Solvent 0 -167 This ) oxidized cytochrome, acetate buffer paH 7_5/glycerol: 111. Cytochrome 2.18pM. (T = ~14 “C; (- - - - -) reduced cytocbrome, T = -26 33; (--) 15 s after addition of oxygen at -26 “C_ The cytockrome was reduced under anaerobic conditiors by addition of 10 ~1 of aqueous sodium dithionite (4 mg/ml).

chrome with simultaneous fast decay

is probably

partial reoxidation

due to the transfer

of cytochrome

of the second

bs [24].

electron

from

This the

other reduced components of the whole systim. In the presence of the substrate ‘7-ethoxycoumarin, hydroxylation can be followed at subzero temperature by the fluorescence increase due to the formation of V-OH coumar& and parallels the decomposition of the oxy-compound, under conditions of a single turnover of the enzyme, since the input of the first electron is blocked under such conditions 1251 (Fig. 4). Tlnis demonstrates clearly the functional role of this compound to initiate the hydroxylation process. The above set of experiments clearly es’tiblishs the optical and functional similarities of oxygenated compounds obtained by an identical procedure (reduction at high temperature, oxygen addition at subzero temperature) with cytochromes P-450 s from various sources and at various integration levels (solubilized or membrane bound). They also confm the role of the ternary complex whereby the substrate and the Fe/O, couple are simultaneously held together as the real starting complex for hydroxylation. (ii) Stability and autoxidation As already indicated, the oxy-ferro compound is not stable even *L the absence of other electron donating agents, and autoxidizes rapidly into the ferric enzyme.

303 s

TABLE 1 Comparison of the spectral from rabbit liver mi axxomes

properties of Ferri, fens, and oxy-ferro cytochrome and from Pseudomow prctidu Compound

Vlavelength &RI-l

Microsod

Pem*

Eacterial umphorfree cytachmme

cm-l)

530 570

llG=*' 12.3 % 0.9 12.3 c 0.7

413 540

82.8 2 2 16.4 2 0-d

420

81.8

558

14.7 t 0.5

417

417

535 569

EacLerial camphorbound cytachrome

Pa

L 5

115 il.6 11.9

408 540

76-7

418 552

70 * 4 J-1 ? 1

15.1

391 510 540

102 13 11.2

408 542

86.5 16

418 552

62 14

*In water/glycerol (111 v/v); buffer 0.1 Tris pz~ 7.5. **At +15 “C ***Taken from published data [18] _ +At -30 “C. **In 5OmM potassium phosphate, pH 7 at 25 q C FlS] _ ++‘In 5OrnM potassium phosphate. pH 7.4, 2OOmM KCl/etbyIene

g!ycol(l:1

v/v)

(19).

In the case of the bacterial cytochrome, this decay is cleanly first order monophasic with the high activation energy of 75 kJ/mol when no subs&&e is bound; the aubxidation occurs also by a monophasic first order reaction, but with a less reproducible rate constant, and leads to a 20 to 30% loss of heme, which is protected by the presence of reagents which are able to react with radicals, such as luminol, methyl viologen, etc. [S] . The a&oxidation mechanism may thus involve some raclicA species eventuaIly able to aCtack the heme ring. On the other hand, a&oxidation of the oxy-ferro compound of microsomal cytochrome is much faster and exhibits two first order phases, differing by a factor of IO in the rate constants. The signifcance of these two

304

~. in n m

i

(~)

M i c r o ~ - o r r e s (3

~

protein /ml )

! = . 3 ~ - C AA

0.01

•

._;~_ ~

~ 420

450

(~)

480

Isolated

"510

P450

A in n m

( 2 iJ~ )

I = - 3 0 "C

Fig. 3. O p t i c a l d i f f e r e n c e s p e c t r a (Fe 2+- 0 2 ) m i n u s ( F e z+) o f c y t o c h r o m e P 4 5 0 (a) R a b b i t liver m i c r o s o m a l s u s p e n s i o n c a . 3 mg p r o t e i n / m l in 0.1M ~ b u f f e r , pH 7.5, plus e t h y l e n e glycol (1:1 v / v ) , ~aH _ 3 5 ° c = 9.9. IT.eduction o f b o t h c u v e t t e s a~ +15 °C by N A D P H (5 × l O - S M ) . All s p e c t r a r e c o r d e d at --35 °C (1) 20 ~ a f t e r a 10 s 0 2 b u b b l i n g ; (2) 3 min 30 s a f t e r 1; (3) a f t e r 5 rain h e a t i n g t o ÷10 °C. (b) I s o l a t e d c y t o c h r o m e P450 f r o m r a b b i t liver in the s a m e s o l v e n t . C y t o c h r o m e c a _ 2~M. R e d u c t i o n at +15 °C by d i t h i o n i t e (5 x 1 0 - S M ) . All s p e c t r a r e c o r d e d at --30 °C (1) 20 a f t e r a 10 s 0 2 b u b b l i n g ; (2) 15 r a i n . a f t e r (1).

p h a s e s is n o t y e t c l e a r ; t h e t r a n s f o r m a t i o n g o e s t h r o u g h p e r c e p t i b l e i s o b e s t i c points. V a r i o u s f a c t o r s s u c h as p a u , t e m p e r a t u r e , t h e pre~cence of l i p i d s , or a substrate, eventually a f f e c t the absolute values of the rate c o n s t a n t , but not t h e b i p h a s i c c h a r a c t e r o f t h e r e a c t i o n . I n p a r t i c u l a r , i n c r e a s i n g t h e P a l l inc r e a s e s t h e s t a b i l i t y o f t h e c o m p o u n d as o b s e r v e d for t h e b a c t e r i a l c y t o c h r o m e [11] _ A c t i v a t i o n e n e r g i e s for the slow p r o c e s s are, h o w e v e r , much l o w e r t h a n in t h e c a s e o f t h e b a c t e r i a l c y t o c h r o m e E a = 4 0 . 1 -+ 4 k J / m o l a t Pall 6 . 2 ( p h o s p h a t e b u f f e r ) , 4 1 . 8 ± 4 k J / m o l a t p a H 7 . 2 ( p h o s p h a t e b u f f e r ) a n d 6 1 . 8 +- 6 k J ! m o l a t P a l l 7 . 9 ( T r i s b u f f e r ) in t h e 1 : 1 ( v / v } H 2 0 / g l y c e r o l mixture.

305 aA

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•

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, = _~o-c

-

,.~

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k\

:

~_~

/

'i

[

(~

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"

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.

-':~---~--'_.--':~r_-

:/

.-

i i "-":

/

-\ •

r

i:

,

~2 ad.dilion

r

!

I

!

r

lime m rain

Pig. 4_ H y d r o x y l a t l o n c o u p l e d t o F e 2 ~ ' - O 2 d e c o m p o s i t i o n in i n t a c t m / c r o s o m e s a t s u b zero t e m p e r a t u r e .

( A ) E v o l u t i o n o f t h e o p t i c a l d i f f e r e n c e s p e c h - ~ . M . i c r o s o r r ~ ! ~ p e a s i o n (3 r a g p ~ o t ~ i n ] m l ) in 4 5 % ( v / v ) e t h y l e n e g l y c o l - - 0 . I M Tris a c e t a t e b u f f e r pa•.2oOc = 8 . 5 , 5 0 p M N A D P H a n d 5 0 0 / ~ m 7 ~ t h o x y c o u ~ r i n . R a t l i n e F e z+ r e c o r d e d a t - - 3 0 °C. Spectra: ( 1 ) 30 s a f t e r o x y g e n b u b b l i n g a~ ~ 3 0 °C; ( 2 ) a f t e r 1 h a t - - 3 0 °C; ( 3 ) a_eter h e a t i n g t o - - 1 0 °C ( 5 m i n ) ; ( g ) h':~olutinn of the f l u o r t ~ c e n c e i n t e n s i t y o f the ~ m e s u ~ i o n ( ~ x c = 385 n m , ~ = 4 6 0 a m ) a f f ~ r o x y g e n a d d i t i o n a t - - 3 0 oC. (a) I n p r e ~ e . ~ c e , ( b ) i n a b s e n c e o£ 7 - e t h o x T c o u n ' t ~ d n . ~ t . r r o ~ ~:l[~.sd;e t h e ~ n . ~ | [ e v e ~ r e c o r d e d a{; t h e r . ~ m e f . e m p ~ r - ~ - ~ a f t e r 5 m~n Co - - 1 0 °C. I n t e n s i t y e x p r ~ s e d in 7 - O H - c o ~ c o n e e n ~ - a t i o n .

(iii) D e c o m p o s i t i o n p r o c e s s M a . R y studies h a v e b e e n d e v o t e d to t h e p r o c e s s b y w h i c h F e 2÷ - 0 2

d e c o m p o s e s i n t o F e ~+ . A n e m i . ~ o n o f l i ~ t w h o s e k i n e t i c s f o l l o w s t h e a u t o x i d a t i o n k i n e t i c s was a~t~ibuted by Sligar to free O~ [ 2 6 ] . We w e r e , h o w e v e r , u n a b l e t o d e t e c t such free s p e c i e s d u r i n g t h e a u t o x i d a t i o n o f the o x y g e n a t ~ i c o m p o u n d o f b a c t e r i a l o r m i c m s o m a l c ~ b r o m e ; t.he m e t h o d s w e used were o x i d a t i o n of n i t r o blue t ~ a z o l i - m , cooxida~ion of epinephrine,

306

0, dependent luminescence of a bacterial luciferase, and the reduction of cytochrome c. Under identical e_xperimental conditions (temperature, pan, solvent), 0; produced during the turnover of xanthine oxidase in the presence of xanthine was easily detected_ Similarly, HzOz was not detected during and after the heat decomposition of this purified complex in the presence or absence of the substrate camphor, while micromolar concentrawere detected_ We must stress that these negative tions of exogenous HzOz results were obtained using the pure chromatographed oxy-ferro complex of bacterial cytochrome, Le., in the absence of the chemical system used to reduce the enzyme, and under conditions of only one turnover of the microsomal compound_ These conditions contrast with those of other authors 127, 281 who obtained some 0; attributable reaction under conditions of continuous electron flow; in those experiments the involvement of the second electron or of more complex reactions producing free 0; cannot be ruled out. Our results are of some importance owing to the possible functional role many authors have attributed to 0, and H2 Oz _ We note that for H, O2 to be formed from free 0, in the medium, dismutation of 2 0, molecules is required_ Such a process would appear highly improbable if 0, is produced in the heme pocket from the direct dissociation Fe2* - O2

++ Fe3+*,

--f Fe3+

+ 0,.

The autosidation process then, seems to occur through a more complex reaction pathway which may eventually involve amino acid residues. Indeed it has been shown that 0, and H,02 cannot be detected in intact organs as cells during drug ~metabolism [ 29]_ The formation of lipid peroxides could be due to secondary processes initiated by ammo acid radicals, or by the electron transferring flavoproteins. Conclusions Now that the pure and stabilized oxy-compounds on the cytochrome P-450 pathway are available, we can study their various decomposition processes in the presence or absence of substrates. Preliminary experiments with the bacterial syste,m show that it cannot accept electrons from methyl viologen or the FMN radical [ 30]_ On the other hand, it decomposes very rapidly in the presence of reduced putidaredoxin via a monophasic first order reaction with k = i-1 X lo- 2 s- ’ at -40 “C [31] _ More steps exist however beyond this reaction and are now being studied in more detail_ The pathway for the second electron in the microsomal system is also under investigation, exploiting subzero temperature methodology with both the intact and reconstituted multi-enzyme complex. Acknowledgements This work was performed with the constant help and interest I. C_ Gunsaius, and Prof. P. Douzou to whom we are most grateful.

of Prof. This

work waz.suppor&d DGRST (contract NaturelIe_

by grant-s from the INSERM (&quipe U-l&S), the no 78.7.0332) md the-Museum National d’Histoire

Referents 1

2 3 4 5 6 ? 8 9 10 11

12 13 14 15 16

17 18 19 20 21 22 23 24 25 26 27

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28 29

30 31

G. D. Nordblom H. Sies, K. Weigl Induction ofDrug 383 - 400. P. Dcbey and C. 1 289 - 1292. G. Hui Bon Hoa. 2 835 - 2 839.

and M. J_ Coon, Arch. Biochem_ Biophys.. 180 (1977) 343 - 347. and C. Waydhas. in R. W. Estabrook and E. Lindenlaub (eds.), The Metabolism. Schattauer Verlag, Stuttgart. New York, 1978, pp. Ezlny,

Biochem.

E. B&gsrd,

Sot.

P_ Debey

Trans_ (577th

Meeting

and I. C. Gunsalus.

OxCord),

Biochemistry.

6 (1979) 17 (1978)