BIOCHIMIE, 1979, 61, 681-687.
Fast photochemical reactions of cytochrome P450 at subzero temperatures. Claude BONFILS, Jean-Louis SALDkNA, Pascale D EBEY, Patrick MAUREL, Claude BALNY and Pierre DOUZOU.
I.N.S.E.R.M. U-128, BP 5051, 3~033 Montpellier c~dex, France.
R~sum~.
Summary.
Fat c o m b i n a n t u n e a c t i v a t i o n r a p i d e p a r l a lumi~re a v e c u n e t h e r m o s t a t i s a t i o n & b a s s e s t e m p 6 r a t u r e s on milieu h y d r o - o r g a n i q u e fluide, on a p u 6tudier d i v e r s e s 6 t a p e s du cycle r6actionnel du c y t o c h r o m e P450. Pour ce faire, u n f l a s h a ~t6 a d a p t 6 sur u n s p e c t r o p h o t o m ~ t r e t y p e A m i n c o - C h a n c e , dont les c u r e s p e u v e n t ~tre t h e r m o s t a t 6 e s & d e s t e m p e r a t u r e s b i e n inf6rieures & 0°C.
Several reactions of the cytochrome P450 multi-step cycle have been studied by iast light
On peut ainsi r6duire le cytochrome P450 ferrique par des photor6ducteurs non sp6cifiques tels que le flavine mononucl~otide r6duit (FMNH2) ou le radical de methyl viologbne (MV ,). On peut alors former le complexe oxyq6n6 Fe2÷-O2 soft p a r addition directe de Foxygene. soft p a r photodissociation de Fe2+-CO en p r e s e n c e d ' o x y q ~ n e . A b a s s e t e m p 6 r a t u r e ce
complexe oxyg6n6 r,e subit plus d'autod6composition et il ne r6aqit pas avec les photor6ducteurs cit6s ci-dessus. Enfin u n e 6tude s y s t 6 m a f i q u e de la recombin a i s o n de C O a p r ~ s photodissociation d u comp l e x e Fe~-CO d u P4~o m i c r o s o m a l purifi6 perm e t de r6v6ler des propri6t~s sp6cifiques de ce c y t o c h r 0 m e p a r c o m p a r a i s o n a v e c ]e cytoc h r o m e bact6rien.
Introduction. The sequential resolution of multi-steps biochemical reactions at subzero temperatures in fluid hydro-organic media is now -well documented [I, 2, 3]. Adaptation of fast techniques (stopped flow [4, 5], flash photolysis [6] and trapping methods [7]) to these particular experimental conditions has opened up a new dimension in the time scale of recording and analysis of the nature and sequence of molecular intermediates [2, 8].
a c t i v a t i o n c o m b i n e d with subzero t e m p e r a tures. A flash device w a s a d a p t e d to a n A m i n c o - C h a n c e DW 2 s p e c t r o p h o t o m e t e r equip e d for subzero t e m p e r a t u r e thermostatisation. The first electron c a n b e introduced into the c y c l e b y non specific r e d u c i n g a g e n t s s u c h a s r e d u c e d flavin m o n o n u c l e o t i d e (FMNH~) or m e t h y l v i o l o q e n r a d i c a l (MV-). This first reduction r e m a i n s a fast p r o c e s s e v e n at subzero t e m p e r a t u r e s . The o x y - c o m p o u n d Fe2+-O2 c a n thus b e f o r m e d either directly from Fe 2÷ or v i a the photodissociation of the carboxy-ferro adduct. Fe2÷-O2 is s t a b l e at subzero t e m p e r a tures t o w a r d s s p o n t a n e o u s autoxidcrtion a s well a s further reduction b y FMNH2 or M W - . In addition, the r e c o m b i n a t i o n of C O after flash photodissociation of Fe2+-CO w a s u s e d to s t u d y in m o r e details the specific b e h a v i o r s of the purified m i c r o s o m a l c y t o c h r o m e . Key words: Cryoenzymologie, Cytochrome P450, Flash photolysis, Photochemistry.
Many systems (single hemoproteins or complex electron transport chains, mitochondrias, chloroplasts) can be activated by light -which provides a clean, rapid and versatile energy source [9, 10, 11]. The triggering light activation processes are quite temperature insensitive, in contrast -with the folio-wing chemical reactions, -which can be drastically slowed down b y lowering the temperature. We have thus used light, combined -with subzero temperature thermostatisation, to study va-
682
C. Bonfils and coll.
v i o u s s t e p s of t h e h y d r o x y l a t i o n c y c l e of h e i n e c o n t a i n i n g c y t o c h r o m e P450 m o n o x y g e n a s e s . T h e s e e n z y m e s f u n c t i o n v i a a n o r d e r e d s e q u e n c e of e v e n t s , i n c l u d i n g s u b s t r a t e b i n d i n g , f e r r i c to f e r rous iron reduction, oxygen binding, and final o x y g e n a c t i v a t i o n b y a s e c o n d e l e c t r o n [12, 13]. T h e c o m p l e x i t y of t h e o v e r a l l r e a c t i o n m a k e s t h e above experimental approach particularly approp r i a t e . T h e s t u d y m a i n l y c o n c e r n s a b a c t e r i a l cy_ t o c h r o m e w-hile s o m e e x p c r i m e n t s w i t h a p u r i f i e d m i c r o s o m a l e n z y m e a r e also i n c l u d e d , a l l o w i n g f o r a c o m p a r i s o n of t h e t w o s y s t e m s .
p o t e n t i a l to switch off a n d the fast dead t i m e of the c o m m u t a t i o n (less t h a n 25 izsec). The device (see figure 1) is placed between the p h o t o m u l t i p l i e r (EMI 9558 B Q) a n d its power supply (provided f r o m the spectrophotometer). The h i g h voltage switch-off is m a d e via a p h o t o t r a n s i s t o r T (MCT2.7316) isolated at 1500 V w h i c h c o m m a n d s a n o t h e r t r a n s i s t o r (BU.105) w o r k i n g in c o m m u t a t i o n . The control of the d u r a t i o n of the cut off, f r o m 0 to 10 msee according to t h e e x p e r i m e n t a l conditions, is m o n i t o r e d b y a m o n o s t a b l e (SN 74122 N) a d j u s t a b l e b y t h e 10 KQ p o t e n t i o m e t e r . (Other values m a y be o b t a i n e d b y m o d i f y i n g t h e monostable-'R-~ circuit). The m o n o s t a b l e is triggered b y a positive f r o n t given b y a SN 7437 circuit associated w i t h a n i n t e r r u p t o r . The flash tubes are fired b y the circuit s h o w n in figure 1, via a n i m p u l s i o n t r a n s f o r m e r w i t h the p r i m a r y circuit fed b y 700 V.
Materials and Methods.
The device provides complete security, t h e flash being fired w h e n the p h o t o m u l t i p l i e r h i g h voltage is effectively switched off.
I. - - F l a s h s g s t e m .
The flash photolysis system used is a modification of a previously described a p p a r a t u s [6] adapted to a n Aminco Chance DW 2 speetrophotometer. The geometry of the cell was modified accordingly. The m o n i t o r i n g b e a m a n d electronic detection system are those of the s p e c t r o p h o t o m e t e r used in dual mode for spectral or kinetic scans.
TI O M Q ~ ' - " to PM c:
BU 105
~,.~/~
F SN
.¢-,.5v
The flash p h o t o l y s i s a p p a r a t u s h a s a long dead t i m e of a b o u t 10 msec. However, the p o s s i b i l i t y of subzero t e m p e r a t u r e s t h e r m o s t a t i s a t i o n largely compensates for t h i s drawback. For example a r e a c t i o n w i t h x 10 msee a t - - 6 0 ° C h a s a t i m e c o n s t a n t a t 20°C of respectively 0.75 msee a n d 0.33 ~see for AH of 4 Keal M-1 a n d 16 Keal M-1.
=
II. - - Products. The b a c t e r i a l cytoehrome P4~o p r e p a r e d in t h e presence of c a m p h o r b y a n a u t o m a t e d modification of t h e procedure of Yu et al. [14] was a generous gift of Prof. I. C. Gunsalus. Liver m i c r o s o m a l eytochrome P~o f r o m p h e n o b a r b i t a l t r e a t e d r a b b i t s was p r e p a r e d according to Coon [15] a n d stored frozen in 20 per cent glyc4fol. P u r e ethylene glycol was f r o m Riedel de Hahn. Other c o m p o u n d s were f r o m Merck. W h e n necessary, aqueous buffers a n d organic solvents were deoxygenated separately b y successive cycles of v a c u u m a n d a r g o n s a t u r a t i o n p r i o r to m i x i n g in 1:1 v o l u m e ratio. Suitable carb o n m o n o x i d e c o n c e n t r a t i o n s were o b t a i n e d b y adding to the s o l u t i o n a defined v o l u m e of w a t e r s a t u r a t e d w i t h CO a t 20°C (solubility 10-3 M).
2
i-.~
i
Results.
SN7437N 22.0v= ~
1. PHOTORF_J)UCTION OF THE FERRIC BACTERIAL CY-
2"2~,v~K O
TOCHROME.
FIO. 1. ~ Electronic device f o r high voltage cut off. (M) m o n o s t a b l e (PM) p h o t o m u l t i p l i e r .
R e d u c t i o n of c y t o c h r o m e P4~o b y a r t i f i c i a l c h e m i c a l r e d u c t o r s s u c h as N a 2 S 2 0 4 is s l o w i n t h e normal temperature range, but can be easily perf o r m e d b y p h o t o a c t i v a t i o n of dyes. T w o r e a g e n t s ~vere u s e d , F M N H 2 d i r e c t l y p h o t o r e d u c e d i n p r e s e n c e of E D T A , a n d t h e MV- r a d i c a l p r o d u c e d b y p h o t o s e n s i t i s a t i o n of a c r i d i n e o r a n g e .
An electronic device allows to switch off the h i g h voltage of the p h o t o m u l t i p l i e r d u r i n g the flash. The m a i n p r o b l e m s in t h i s o p e r a t i o n are the h i g h negative
At r o o m t e m p e r a t u r e F M N H 2 r e a c t s v e r y s l o w l y w i t h t h e t r a c e a m o u n t s of o x y g e n e v e n t u a l l y c o n ruminating the solution even after the extensive a r g o n a e r a t i o n (see M a t e r i a l s ) [16]. T h e o x i d a t i o n
to flash
~ I
I~F
T
BIOCHIMIE, 1979, 61, n ° 5-6.
C.tltochrome P,,ao photochemistr~I. rate b e c o m e s negligible at l o w t e m p e r a t u r e s a n d it does not i n t e r f e r e w i t h the r e d u c t i o n of c y t o c h r o me P~s0. M i c r o m o l a r c o n c e n t r a t i o n s of FeZ+ : R H are r e d u c e d by excess FMNH 2 t h r o u g h a pseudofirst o r d e r m o n o p h a s i c process. The s e c o n d o r d e r A'
® ~//,~.
-, ~ ~./-__
,,tf'~'~'~;"
Fe 3+. RH
. . . . Fe 2+, RH
0,05
"J
f 85
400
415
30
445
460 ~. in nm
'~0.02
o/~ -0.02
-0.06i -0.06i
5
10
15 time in scc
FIG. 2. - - Bacterial cytochro~te P~5o : reduction bg MV. radical. Solvent 1:1 (v/v) mixture of 100 mM
Na+/K+ phosphate buffer pH 7.3 and ethylene glycol (pa~ -= 7.8 at 20°C), containing 5 mM EDTA, 280 mM camphor, 85 IxM MY2+, 36 t~M acridine orange. Cytochrome 4.2 ~M. Optical spectra ( -) before (Fe3+.RH) and (. . . . ) after (Fe2+.RH) photoreduction at respectively (~) 7°C and (if)--29°C. Dual mode spectral scan. Reference wavelength 404 nm. Kinetics of heme reduction after a flash, hAso2_~ at 7°C (~) and - - 29°C (~).
rate constant is 3.9 ± 0.2 10 a M-1 s ec-Z at 4°C ; its A r r h e n i u s plot is l i n e a r b e f w e e n 4°C and - - 30°C w i t h an a c t i v a t i o n e n e r g y of 6.8 ± 0.2 Kcal M-1. In contrast, the MV. r a d i c a l reacts w i t h traces of oxygen w i t h i n the dead t i m e of the flash (k = 7.7 l0 s M -1 sec-1) [17J. U n d e r our e x p e r i m e n t a l c o n d i t i o n s 3 to 5 t~M of MV ° are p r o d u c e d at each flash, d e p e n d i n g on the voltage of discharge. W h e n c y t o c h r o m e P450 is p r e s e n t in c o m p a r a b l e c o n c e n t r a t i o n , MV. decays via a second process
BIOCHIMIE, 1979, 61, n ° 5-6.
w h i c h is a c c o m p a n i e d by r e d u c t i o n of the h e m e as j u d g e d from the a b s o r b a n c e d e c r e a s e b e t w e e n 392 nm ( m a x i m u m of the mostly h i g h spin Fez +. RH adduct) and 404 nm (isobestic b e t w e e n Fea+. RH). I n i t i a l MV- c o n c e n t r a t i o n s are o b t a i n e d from the initial i n c r e a s e in a b s o r b a n c e (Ae392.404 = 15.9 mM-1). Second o r d e r plots w e r e c o m p u t e d using Ae392.404(Fez + . RH ~ Fe '2÷. RH) = 27 mM -1 for the h i g h spin f e r r i c c y t o c h r o m e and Aea92_404 (Fez + ~ Fe 2+) = 11.5 raM-1 for t h e l o w spin f o r m of the c y t o c h r o m e . T h e e q u i l i b r i u m b e t w e e n high and l o w spin, w h i c h d e p e n d s on the e x p e r i m e n t a l c o n d i t i o n s [18], w a s taken f r o m t h e o p t i c a l spect r u m m e a s u r e d at each t e m p e r a t u r e (figure 2). T h e s e c o n d o r d e r rate constant is 1.6 105 M-1 sec-1 at 7°C in a h y d r o - o r g a n i c m e d i u m , w i t h an activ a t i o n energy of 4.5 _ 0.5 Kcal M-1 b e t w e e n + 7°C and - - 30°C. This l o w value is n o t e w o r t h y since t h e o t h e r steps of the r e a c t i o n cycle h a v e a c t i v a t i o n energies in the 8-12 Kcal M-1 range. Thus, the p h o t o c h e m i c a l i n p u t of l h e first electron of t h e r e a c t i o n cycle r e m a i n s a v e r y fast process, even at l o w t e m p e r a t u r e . It shoutd be noted that the o x y - f e r r o c y t o c h r o me (Fe ~+. RH), w h i c h is stable for s e v e r a l hours, is r e a d i l y f o r m e d at subzero t e m p e r a t u r e s by the a d d i t i o n of 0 2 to the p h o t o r e d u c e d c y t o c h r o m e [19].
t
t Flash
683
T h e m i c r o s o m a l m e m b r a n e b o u n d or solubilized c y t o c h r o m e 'P450 c a n be p h o t o r e d u c e d in a sim i l a r m a n n e r by the t w o above d e s c r i b e d systems (MV. has even been s h o w n to be a n e c e s s a r y m e d i a t o r for the e l e c t r o n t r a n s f e r b e t w e e n dit h i o n i t e and f e r r i c c y t o c h r o m e [20]). W h e n oxygen is b u b b l e d t h r o u g h such a p h o t o c h e m i c a l l y r e d u c e d a n a e r o b i c solution at subzero t e m p e r a t u res, it consumes q u i c k l y the p h o t o r e d u c e d species. At t h e same time ~the oxygen b i n d s to Fe 2+ to f o r m an o x y - f e r r o c o m p o u n d , w h i c h is stable at - - 30°C and h i g h pa H (C. Bonfils, P. Maurel, P. Debey, to be published). In contrast, w h e n Na2S20 4 is used as a c h e m i c a l r e d u c t o r only, it reacts v e r y s l o w l y at subzero t e m p e r a t u r e s w i t h a d d e d oxygen and the r e m a i n i n g excess destroys r a p i d l y the oxyferro compound.
2.
PHOTODISSOCIATION
OF
THE
CARBOXY - F~BBO
COMPOUND.
As an a l t e r n a t i v e to the above, light can be used to i n t r o d u c e a r a p i d p e r t u r b a t i o n in a system p r e v i o u s l y in e q u i l i b r i u m . At low t e m p e r a t u r e s the subsequent r e t u r n to e q u i l i b r i u m is dras-
C. B o n f i l s a n d coll.
684
tieally slowed down, thus a l l o w i n g i n t e r m e d i a t e species to be t r a p p e d a n d characterized by r a p i d optical s c a n n i n g ,
c o m p o u n d at a n y t e m p e r a t u r e a n d CO c o n c e n t r a tion from 15 ~M to 80 ~M can be expressed as a m i x t u r e of two first o r d e r phases of n e a r l y iden% Photoreduction
k in sec -1
t
®
10 6
10C
kf
/
50
®
10 5
z.~O'
;.4
'
;.6
'
;.8 " 1~( 0K-1 )
0
50
~
50
-~o Temperature -'30 ~
FIG. 3. - Arrhenitts plot o f the fast (kt) arts slow (k~) rate consrants for recombination of CO to microsomal cytochrome P~so after flash photolysis. Solvent 50/50 (v/v) mixture of glycerol and 100 mM phosphate buffer pH 7.5. Temperature dependence of the photodissofiation yield 10 msec after the flash. (~ Solvent contains only 10 per cent (v/v) glycerol. (~ Solvent contains 50 per cent (v/v) glycerol.
a) Bacterial c y t o c h r o m e . R e c o m b i n a t i o n of the heme ligand w i t h CO after flash p h o t o d i s s o c i a t i o n of Fe2÷-CO b o n d has been studied over a wide range of subzero temperates. W i t h the bacterial cytochrome, the rec o m b i n a t i o n is first o r d e r m o n o p h a s i c at all temp e r a t u r e s studied, w i t h a rate constant of 3.5 104 M-1 see-1 at 12°C (i.e. very close to the value measured i n aqueous buffer at 4°C [21] 3.8 104 M-1 sec-1) a n d an activation energy of 8 -4- 0.4 Kcal M-1 in h y d r o - o r g a n i c medium. b) Microsomal c y t o c h r o m e . We have s i m i l a r l y studied the p h o t o d i s s o c i a t i o n of the carboxy-ferro complex of purified microsomal cytochrome P45o in glycerol-aqueous mixtures. Glycerol was chosen for its better p r o t e c t i n g effect on the m i c r o s o m a l cytochrome. The results are far more complex t h a n w i t h the bacterial cytochrome. The k i n e t i c s of the observed r e c o m b i n a t i o n after flash p h o t o d i s s o c i a l i o n of the carboxy-ferro
BIOCHIMIE, 1979, 61, n ° 5-6.
tical amplitude. The first phase is c.a. 6 to 8 times faster t h a n the last one, this ratio r e m a i n i n g constant w i t h temperature. The A r r h e n i u s plots of the two observed rate constants (k~ a n d k 0 are l i n e a r b e t w e e n + 20°C a n d - - 30°C w i t h an i d e n t i c a l activation energy of 11.5 -4- 1.5 K c a l / m o l e (figure 3). kf a n d k~ increase both p r o p o r t i o n a l l y to CO c o n c e n t r a t i o n up to 80 ~M. F i n a l l y w h e n the c o n c e n t r a t i o n of CO-free cytochrome is extrapolated to the time t = 0, only 70 per cent to 50 per cent p h o t o d i s s o c i a t i o n is f o u n d at + 20°C i n 50 per cent glycerol. This yield varies s o m e w h a t w i t h the p r e p a r a t i o n but r e m a i n s m u c h less t h a n 100 per cent i n p r e s e n c e of 50 per cent glycerol. It does not v a r y w i t h the CO c o n c e n t r a t i o n nor w i t h the flash power, n o r w i t h the p r e s e n c e of the detergent Renex (0.1 per cent or 0.5 per cent). W h e n t e m p e r a t u r e decreases the i n i t i a l p h o t o d i s s o c i a t i o n increases up to a m a x i m u m at c.a. - - 15°C -4- 3°C. I n presence of only 10 per cent glycerol, the r e c o m b i n a t i o n r e m a i n s b i p h a s i c but the i n i t i a l p h o t o d i s s o c i a t i o n yield is much higher (90 per
C y t o c h r o m e P~5o p h o t o c h e m i s t r y . cent to 100 per cent) a n d decreases slowly w h e n t e m p e r a t u r e decreases from 20°C to - - 8°C. Although the a m p l i t u d e a n d ratio of the two r e c o m b i n a t i o n phases a n d the i n i t i a l photodissoclarion yield v a r y q u a n t i t a t i v e l y somewhat ~vith the sample a n d its history, even ~vhen starting from the same stock solution, the basic features described above r e m a i n qualitatively u n c h a n g e d . F u r t h e r m o r e we checked in each case that the cytochrome was saturated w i t h CO and the soIution essentially free of P420.
685
boxy-ferro c o m p o u n d is too slow to allow spontaneous d i s p l a c e m e n t of CO b y 0 2 via : Fe 2÷ - -
kl CO ~- Fe 2* -[- CO k. 1
k2 Fe2+ + 02 ~ Fe 2 + _
(1)
02
(2)
k_2
However, 02 b i n d i n g is m u c h faster t h a n CO b i n d i n g . F u r t h e r m o r e , Fe2* __ 02 is only p o o r l y
3. FORMATION OF THE OXY-FERRO COMPOUND, Light p h o t o d i s s o c i a t i o n of m u l t i p l e ligand equil i b r i a alIows to form easily the oxy-ferro com-
i
0.05
t = .30°C
I
0.05
x
\ \ \
i
t
t
i
I
I
T
370
385
400
415
430
/=,45
i
460 L
in
nm
FIG. 4. - - Formation of the oxy-eomplex of bacterial cytochrome P~o after photodissociation of FeSc+o" RH in presence of 0~. Solvent 1:1 (v/v) mixture of 100 mM Na*/K+ phosphate buffer pH 7.3 and ethylene glycol (pare: 7.8 at 20°C) containing 280 ~tM camphor. Cytochrome P~5o, 6.16 ~tM ; CO. 20 ~tM ; t : 30°C ; wavelength interval 90 nm, speed 20 nm.sec-1. Reference wavelength 404 nm. (~) Fe~+'RH spectrum. (~) after 5 sec aeration with 0~. (~) after flash.
p o u n d from the carboxy-ferro c o m p o u n d . At - - 30°C the dissociation rate c o n s t a n t of the car-
BIOCHIMIE, 1979, 61, n ° 5-6.
i 380
i
i 400
i 420
i 440
\
i
t
k
;~ I n n m
Fro. 5 . - Spectral intermediates of bacterial cytochrome P~so at --20°C, (~) after flash photoreduction by FMNI-Lj (Fe~+RH), (~) after 02 bubbling (Fe~+'RH), (~) and (~ after several flashes on the same oxygenated solution. Solvent as in figure 2 with 5 mM EDTA, 300 ttM camphor, 6 ttM cytochrome (final concentrations). Dual mode spectral scan. Reference wavelength 404 nm.
photodissociable ; a c c o r d i n g l y flash photodissoclarion ~t - - 30°C of Fe 2÷ - - CO in the presence of 20 ~M CO and 100 ~M 02 (figure 4) results in the f o r m a t i o n of 100 p e r cent F e 2÷ - - 02 w i t h i n 10 msec. It is n o t e w o r t h y that at - - 30°C, Fe - R H is stable t o w a r d s a u t o x i d a t i o n (1/2 t ---- 8 hours) but not completely t o w a r d s l i g a n d exchange (slow d i s p l a c e m e n t of 02 b y CO).
C. BonfiIs and coll.
686 4. I N T E R A C T I O N
OF
THE
OXY-FERRO
CYTOCHROME
W I T H REI)UCING RADICALS.
A solution of the oxy-forro adduct p r e p a r e d in presence of a photoactive system (FMN or AO % l~fV) a n d stabilized at subzero temperatures, can be i r r a d i a t e d to p r o d u c e r e d u c i n g radicals. As s h o w n b y figure 5, successive flashes at - - 3 0 ° C on a Fe 2. - - 0 2 solution c o n t a i n i n g FMN p r o d u c e a progressive shift of the Sorer m a x i m u m from 418 (Fe~+ . RH) to 410 n m (Fe 2+ . RH). The n u m b e r of flashes (i.e. the total d u r a t i o n of illumin.ation) necessary for complete t r a n s f o r m a t i o n depends only on the oxygen c o n c e n t r a t i o n , all other conditions b e i n g identical. This clearly shows that the ouly effect of p h o t o r e d u c e d radicals is to consume oxygen progressively a n d shift the equilikl b r i u m Fe 2+. R,tI -t- 0~. ~ F e ~ . RH t o w a r d s re-
k.1 duction. After complete t r a n s f o r m a t i o n , a second oxygen b u b b l i n g restores the 418 n m b a n d . I d e n t i c a l resutts were o b t a i n e d w i t h the AO -F MV system. Thus n e i t h e r FMNH., nor FMNH 2 or MV- are able to t r a n s f e r the second electron to the oxy-ferro c o m p o u n d .
Discussion. P h o t o c h e m i c a l systems are very useful for studies on the first electron r e d u c t i o n of c y t o c h r o m e P450, since they p r o v i d e fast electron i n p u t even at low temperatures. It was quite s u r p r i s i n g however to observe that electrons p r o d u c e d in this m a n n e r were u n a b l e to react w i t h the oxygenated c o m p o u n d . I n the n a t u r a l h y d r o x y l a t i o n cycle addition of the second electron results in oxygen activation. These observations contrast also with the h i g h reactivity of the same r e d u c i n g agents w i t h formal analogs of oxygenated eytoehrome P45o, such as c o m p o u n d III of peroxydases [22]. These data emphasize the u n i q u e c h a r a c t e r of c y t o c h r o m e P45o a n d the definite r e q u i r e m e n t of a specific electron donating-effector p r o t e i n for the second electron r e d u c t i o n of the oxygenated complex [13]. Although not directly related to the enzymatic cycle, the k i n e t i c s and t h e r m o d y n a m i c s of carb o n m o n o x i d e b i n d i n g to the ferrous i r o n can be used to characterize the heme center. F o r the bacterial cytochrome, lhe r e c o m b i n a t i o n after flash photolysis of Fe2÷_ CO follows a simple first order r e a c t i o n in m i x e d solvent at a n y tempera-
BIOCHIMIE, 1979,
61, n ° 5-6.
ture above its freezing point, and provides a test of the i n o c u i t y of the o r g a n i c solvent. The u n i q u e features observed by flash photolysis of the purified nlierosomal cytochrome P450 are w o r t h to m e n t i o n a n d discuss. Similar results were already o b t a i n e d w i t h ~ h o l e microsomes or c r u d e p r e p a r a t i o n s of solubilized e y t o e h r o m e [6]. The two phases could h a r d l y r e p r e s e n t two pop u l a t i o n s of cytoehrome P4no since the preparation is electrophoreticaliy pure and, on the other h a n d , the h i g h l y heterogeneous m i e r o s o m a l population exhibits the same p r o p o r t i o n of fast and slow r e a c t i n g material [6]. F o r the same reason, they c a n n o t be linked to different p h y s i c a l (memb r a n e b o u n d or soluble) or a g g r e g a t i o n state, since Renex has no substantial effect. They could r a t h e r rely on different possible c o n f o r m a t i o n s of the molecule, r e l a x i n g on the 10 .2 sec to I sec time scale i n the -J- 20 ° - - 30°C t e m p e r a t u r e range. The i n c o m p l e t e a n d t e m p e r a t u r e - d e p e n d e n t (( i n i t i a l >> p h o t o d i s s o c i a t i o n c a n n o t he due to a low q u a n t u m yield since 100 per cent photodissociation is obtained w i t h 10 per cent glycerol. It r a t h e r i n d i c a t e s a t h i r d r a p i d phase o c c u r r i n g dur i n g the dead time (10 msec) of the method, similar to the process I ( r e c o m b i n a t i o n i n the pocket w i t h a cage effect) a l r e a d y observed by Austin et al. [10, 23] w i t h other soluble h e m o p r o l e i n s (myoglobin, hemoglobin, bacterial c y t o c h r o m e P450) at m u c h lower t e m p e r a t u r e s ( ( 2 0 0 ° K ) in frozen or glassy state. T h e i r photolysis experiments extended to the n a n o - s e c o n d range and cryogenic t e m p e r a t u r e s allowed to resolve the rec o m b i n a t i o n of a gazeous ligand in various elemen.tary processes a n d can serve as a basis for i n t e r p r e t i n g our present results on a more limited time and t e m p e r a t u r e scale. P a r t i c u l a r l y the glycerol a n d t e m p e r a t u r e effect on the yield could be thought out as a c o m p e t i t i o n between microviseosity a n d t e m p e r a t u r e effect on rate constants. The fact that two rate constants are observable at room t e m p e r a t u r e seems to i n d i c a t e that the mol e c u l a r r e l a x a t i o n b e t w e e n various c o n f o r m a t i o n s are very slow as c o m p a r e d w i t h other hemoproreins [10]. It w o u l d be of interest to evaluate the relevance of these p h y s i c o - e h e m i e a l proterties to the u n u s u a l properties of the liver h y d r o x y l a s e as c o m p a r e d to other c y t o c h r o m e P450 systems, i.e. its heterogeneity [24], a n d b r o a d specificity [25]. F r o m a more p r a c t i c a l p o i n t of view, c o m b i n a tion of high activation a n d cooling-heating cycles m a y overcome one of the major draw backs of the subzero t e m p e r a t u r e method, i.e. the high v i s e o -
Cytochrome P ~5o photochemistry. sity which sometimes prevents a proper mixing of r e a c t a n t s . T h e v a r i o u s p o s s i b i l i t i e s of t h i s m e t h o d are n o w b e i n g tested on other sties of /he c y t o c h r o m e P45o c y c l e a n d o t h e r m u l t i - c o m p o nents electron transport chains.
Acknowledgements. This w o r k w a s s u p p o r t e d by grants f r o m the l n s t i t u t N a t i o n a l de la Santd et de la Recherche Mddicale (U-128), f r o m the C.N.R.S., f r o m the D.G.R.S.T. (contrat n ° 7870332) and f r o m the F o n d a t i o n p o u r la Recherche M~dicale F r a n f a i s e .
REFERENCES. 1. Douzou P. (1977) Adv. in E n z y m o l . , 45, 157-272. 2. Douzou, P. (1977) , Acad. Press, London. 3. Cox, R. P. (1978) Biochem. Bey., 6, 689-697. 4. Hut Bon Hoa, G. & Douzou, P. (1972) Anal. Biochem., 51, 127-136. 5. Ballou, D. (1971) Ph. D. Thesis, University of Michigan, Ann Arbor, Michigan, U.S.A. 6. Debey, P., Balny, C. ~ Douzou, P. (1973) F E B S Letters. 35, 86-90. 7. Chance, B., Graham, N. ,~ Legallais~ V. (1975) Anal. Biochem., 67, 552-579. 8. Douzou, P., Sireix, R. ~ Travers, F. (1970) Proc. Natl. Acad. Sci. U.S.A., 66, 787-792. 9. Chance, B., Saronio, C. ~ Leigh, J. S. Jr. (1975) Proc. Natl. Acad. Sci. U.S.A., 72, 1635-1640.
BIOCHIMIE, 1979, 61, n ° 5-6.
687
10. Austin, R. H., Beeson_ K. W., Eisenstein, L., F r a u e n felder, H. ,~ Gunsalus, I. C. (1975) B i o c h e m i s t r y , 14, 5355-5373. 11. Cox, R. (1975) B i o e h i m . B i o p h y s . Aeta, 387, 588-598. 12. Peterson, J. A., Ishimura, Y., Baron, J. ~ Estabrook, R. W. (1973) in
Fast photochemical reactions of cytochrome P450 at subzero temperatures. Claude BONFILS, Jean-Louis SALDkNA, Pascale D EBEY, Patrick MAUREL, Claude BALNY and Pierre DOUZOU.
I.N.S.E.R.M. U-128, BP 5051, 3~033 Montpellier c~dex, France.
R~sum~.
Summary.
Fat c o m b i n a n t u n e a c t i v a t i o n r a p i d e p a r l a lumi~re a v e c u n e t h e r m o s t a t i s a t i o n & b a s s e s t e m p 6 r a t u r e s on milieu h y d r o - o r g a n i q u e fluide, on a p u 6tudier d i v e r s e s 6 t a p e s du cycle r6actionnel du c y t o c h r o m e P450. Pour ce faire, u n f l a s h a ~t6 a d a p t 6 sur u n s p e c t r o p h o t o m ~ t r e t y p e A m i n c o - C h a n c e , dont les c u r e s p e u v e n t ~tre t h e r m o s t a t 6 e s & d e s t e m p e r a t u r e s b i e n inf6rieures & 0°C.
Several reactions of the cytochrome P450 multi-step cycle have been studied by iast light
On peut ainsi r6duire le cytochrome P450 ferrique par des photor6ducteurs non sp6cifiques tels que le flavine mononucl~otide r6duit (FMNH2) ou le radical de methyl viologbne (MV ,). On peut alors former le complexe oxyq6n6 Fe2÷-O2 soft p a r addition directe de Foxygene. soft p a r photodissociation de Fe2+-CO en p r e s e n c e d ' o x y q ~ n e . A b a s s e t e m p 6 r a t u r e ce
complexe oxyg6n6 r,e subit plus d'autod6composition et il ne r6aqit pas avec les photor6ducteurs cit6s ci-dessus. Enfin u n e 6tude s y s t 6 m a f i q u e de la recombin a i s o n de C O a p r ~ s photodissociation d u comp l e x e Fe~-CO d u P4~o m i c r o s o m a l purifi6 perm e t de r6v6ler des propri6t~s sp6cifiques de ce c y t o c h r 0 m e p a r c o m p a r a i s o n a v e c ]e cytoc h r o m e bact6rien.
Introduction. The sequential resolution of multi-steps biochemical reactions at subzero temperatures in fluid hydro-organic media is now -well documented [I, 2, 3]. Adaptation of fast techniques (stopped flow [4, 5], flash photolysis [6] and trapping methods [7]) to these particular experimental conditions has opened up a new dimension in the time scale of recording and analysis of the nature and sequence of molecular intermediates [2, 8].
a c t i v a t i o n c o m b i n e d with subzero t e m p e r a tures. A flash device w a s a d a p t e d to a n A m i n c o - C h a n c e DW 2 s p e c t r o p h o t o m e t e r equip e d for subzero t e m p e r a t u r e thermostatisation. The first electron c a n b e introduced into the c y c l e b y non specific r e d u c i n g a g e n t s s u c h a s r e d u c e d flavin m o n o n u c l e o t i d e (FMNH~) or m e t h y l v i o l o q e n r a d i c a l (MV-). This first reduction r e m a i n s a fast p r o c e s s e v e n at subzero t e m p e r a t u r e s . The o x y - c o m p o u n d Fe2+-O2 c a n thus b e f o r m e d either directly from Fe 2÷ or v i a the photodissociation of the carboxy-ferro adduct. Fe2÷-O2 is s t a b l e at subzero t e m p e r a tures t o w a r d s s p o n t a n e o u s autoxidcrtion a s well a s further reduction b y FMNH2 or M W - . In addition, the r e c o m b i n a t i o n of C O after flash photodissociation of Fe2+-CO w a s u s e d to s t u d y in m o r e details the specific b e h a v i o r s of the purified m i c r o s o m a l c y t o c h r o m e . Key words: Cryoenzymologie, Cytochrome P450, Flash photolysis, Photochemistry.
Many systems (single hemoproteins or complex electron transport chains, mitochondrias, chloroplasts) can be activated by light -which provides a clean, rapid and versatile energy source [9, 10, 11]. The triggering light activation processes are quite temperature insensitive, in contrast -with the folio-wing chemical reactions, -which can be drastically slowed down b y lowering the temperature. We have thus used light, combined -with subzero temperature thermostatisation, to study va-
682
C. Bonfils and coll.
v i o u s s t e p s of t h e h y d r o x y l a t i o n c y c l e of h e i n e c o n t a i n i n g c y t o c h r o m e P450 m o n o x y g e n a s e s . T h e s e e n z y m e s f u n c t i o n v i a a n o r d e r e d s e q u e n c e of e v e n t s , i n c l u d i n g s u b s t r a t e b i n d i n g , f e r r i c to f e r rous iron reduction, oxygen binding, and final o x y g e n a c t i v a t i o n b y a s e c o n d e l e c t r o n [12, 13]. T h e c o m p l e x i t y of t h e o v e r a l l r e a c t i o n m a k e s t h e above experimental approach particularly approp r i a t e . T h e s t u d y m a i n l y c o n c e r n s a b a c t e r i a l cy_ t o c h r o m e w-hile s o m e e x p c r i m e n t s w i t h a p u r i f i e d m i c r o s o m a l e n z y m e a r e also i n c l u d e d , a l l o w i n g f o r a c o m p a r i s o n of t h e t w o s y s t e m s .
p o t e n t i a l to switch off a n d the fast dead t i m e of the c o m m u t a t i o n (less t h a n 25 izsec). The device (see figure 1) is placed between the p h o t o m u l t i p l i e r (EMI 9558 B Q) a n d its power supply (provided f r o m the spectrophotometer). The h i g h voltage switch-off is m a d e via a p h o t o t r a n s i s t o r T (MCT2.7316) isolated at 1500 V w h i c h c o m m a n d s a n o t h e r t r a n s i s t o r (BU.105) w o r k i n g in c o m m u t a t i o n . The control of the d u r a t i o n of the cut off, f r o m 0 to 10 msee according to t h e e x p e r i m e n t a l conditions, is m o n i t o r e d b y a m o n o s t a b l e (SN 74122 N) a d j u s t a b l e b y t h e 10 KQ p o t e n t i o m e t e r . (Other values m a y be o b t a i n e d b y m o d i f y i n g t h e monostable-'R-~ circuit). The m o n o s t a b l e is triggered b y a positive f r o n t given b y a SN 7437 circuit associated w i t h a n i n t e r r u p t o r . The flash tubes are fired b y the circuit s h o w n in figure 1, via a n i m p u l s i o n t r a n s f o r m e r w i t h the p r i m a r y circuit fed b y 700 V.
Materials and Methods.
The device provides complete security, t h e flash being fired w h e n the p h o t o m u l t i p l i e r h i g h voltage is effectively switched off.
I. - - F l a s h s g s t e m .
The flash photolysis system used is a modification of a previously described a p p a r a t u s [6] adapted to a n Aminco Chance DW 2 speetrophotometer. The geometry of the cell was modified accordingly. The m o n i t o r i n g b e a m a n d electronic detection system are those of the s p e c t r o p h o t o m e t e r used in dual mode for spectral or kinetic scans.
TI O M Q ~ ' - " to PM c:
BU 105
~,.~/~
F SN
.¢-,.5v
The flash p h o t o l y s i s a p p a r a t u s h a s a long dead t i m e of a b o u t 10 msec. However, the p o s s i b i l i t y of subzero t e m p e r a t u r e s t h e r m o s t a t i s a t i o n largely compensates for t h i s drawback. For example a r e a c t i o n w i t h x 10 msee a t - - 6 0 ° C h a s a t i m e c o n s t a n t a t 20°C of respectively 0.75 msee a n d 0.33 ~see for AH of 4 Keal M-1 a n d 16 Keal M-1.
=
II. - - Products. The b a c t e r i a l cytoehrome P4~o p r e p a r e d in t h e presence of c a m p h o r b y a n a u t o m a t e d modification of t h e procedure of Yu et al. [14] was a generous gift of Prof. I. C. Gunsalus. Liver m i c r o s o m a l eytochrome P~o f r o m p h e n o b a r b i t a l t r e a t e d r a b b i t s was p r e p a r e d according to Coon [15] a n d stored frozen in 20 per cent glyc4fol. P u r e ethylene glycol was f r o m Riedel de Hahn. Other c o m p o u n d s were f r o m Merck. W h e n necessary, aqueous buffers a n d organic solvents were deoxygenated separately b y successive cycles of v a c u u m a n d a r g o n s a t u r a t i o n p r i o r to m i x i n g in 1:1 v o l u m e ratio. Suitable carb o n m o n o x i d e c o n c e n t r a t i o n s were o b t a i n e d b y adding to the s o l u t i o n a defined v o l u m e of w a t e r s a t u r a t e d w i t h CO a t 20°C (solubility 10-3 M).
2
i-.~
i
Results.
SN7437N 22.0v= ~
1. PHOTORF_J)UCTION OF THE FERRIC BACTERIAL CY-
2"2~,v~K O
TOCHROME.
FIO. 1. ~ Electronic device f o r high voltage cut off. (M) m o n o s t a b l e (PM) p h o t o m u l t i p l i e r .
R e d u c t i o n of c y t o c h r o m e P4~o b y a r t i f i c i a l c h e m i c a l r e d u c t o r s s u c h as N a 2 S 2 0 4 is s l o w i n t h e normal temperature range, but can be easily perf o r m e d b y p h o t o a c t i v a t i o n of dyes. T w o r e a g e n t s ~vere u s e d , F M N H 2 d i r e c t l y p h o t o r e d u c e d i n p r e s e n c e of E D T A , a n d t h e MV- r a d i c a l p r o d u c e d b y p h o t o s e n s i t i s a t i o n of a c r i d i n e o r a n g e .
An electronic device allows to switch off the h i g h voltage of the p h o t o m u l t i p l i e r d u r i n g the flash. The m a i n p r o b l e m s in t h i s o p e r a t i o n are the h i g h negative
At r o o m t e m p e r a t u r e F M N H 2 r e a c t s v e r y s l o w l y w i t h t h e t r a c e a m o u n t s of o x y g e n e v e n t u a l l y c o n ruminating the solution even after the extensive a r g o n a e r a t i o n (see M a t e r i a l s ) [16]. T h e o x i d a t i o n
to flash
~ I
I~F
T
BIOCHIMIE, 1979, 61, n ° 5-6.
C.tltochrome P,,ao photochemistr~I. rate b e c o m e s negligible at l o w t e m p e r a t u r e s a n d it does not i n t e r f e r e w i t h the r e d u c t i o n of c y t o c h r o me P~s0. M i c r o m o l a r c o n c e n t r a t i o n s of FeZ+ : R H are r e d u c e d by excess FMNH 2 t h r o u g h a pseudofirst o r d e r m o n o p h a s i c process. The s e c o n d o r d e r A'
® ~//,~.
-, ~ ~./-__
,,tf'~'~'~;"
Fe 3+. RH
. . . . Fe 2+, RH
0,05
"J
f 85
400
415
30
445
460 ~. in nm
'~0.02
o/~ -0.02
-0.06i -0.06i
5
10
15 time in scc
FIG. 2. - - Bacterial cytochro~te P~5o : reduction bg MV. radical. Solvent 1:1 (v/v) mixture of 100 mM
Na+/K+ phosphate buffer pH 7.3 and ethylene glycol (pa~ -= 7.8 at 20°C), containing 5 mM EDTA, 280 mM camphor, 85 IxM MY2+, 36 t~M acridine orange. Cytochrome 4.2 ~M. Optical spectra ( -) before (Fe3+.RH) and (. . . . ) after (Fe2+.RH) photoreduction at respectively (~) 7°C and (if)--29°C. Dual mode spectral scan. Reference wavelength 404 nm. Kinetics of heme reduction after a flash, hAso2_~ at 7°C (~) and - - 29°C (~).
rate constant is 3.9 ± 0.2 10 a M-1 s ec-Z at 4°C ; its A r r h e n i u s plot is l i n e a r b e f w e e n 4°C and - - 30°C w i t h an a c t i v a t i o n e n e r g y of 6.8 ± 0.2 Kcal M-1. In contrast, the MV. r a d i c a l reacts w i t h traces of oxygen w i t h i n the dead t i m e of the flash (k = 7.7 l0 s M -1 sec-1) [17J. U n d e r our e x p e r i m e n t a l c o n d i t i o n s 3 to 5 t~M of MV ° are p r o d u c e d at each flash, d e p e n d i n g on the voltage of discharge. W h e n c y t o c h r o m e P450 is p r e s e n t in c o m p a r a b l e c o n c e n t r a t i o n , MV. decays via a second process
BIOCHIMIE, 1979, 61, n ° 5-6.
w h i c h is a c c o m p a n i e d by r e d u c t i o n of the h e m e as j u d g e d from the a b s o r b a n c e d e c r e a s e b e t w e e n 392 nm ( m a x i m u m of the mostly h i g h spin Fez +. RH adduct) and 404 nm (isobestic b e t w e e n Fea+. RH). I n i t i a l MV- c o n c e n t r a t i o n s are o b t a i n e d from the initial i n c r e a s e in a b s o r b a n c e (Ae392.404 = 15.9 mM-1). Second o r d e r plots w e r e c o m p u t e d using Ae392.404(Fez + . RH ~ Fe '2÷. RH) = 27 mM -1 for the h i g h spin f e r r i c c y t o c h r o m e and Aea92_404 (Fez + ~ Fe 2+) = 11.5 raM-1 for t h e l o w spin f o r m of the c y t o c h r o m e . T h e e q u i l i b r i u m b e t w e e n high and l o w spin, w h i c h d e p e n d s on the e x p e r i m e n t a l c o n d i t i o n s [18], w a s taken f r o m t h e o p t i c a l spect r u m m e a s u r e d at each t e m p e r a t u r e (figure 2). T h e s e c o n d o r d e r rate constant is 1.6 105 M-1 sec-1 at 7°C in a h y d r o - o r g a n i c m e d i u m , w i t h an activ a t i o n energy of 4.5 _ 0.5 Kcal M-1 b e t w e e n + 7°C and - - 30°C. This l o w value is n o t e w o r t h y since t h e o t h e r steps of the r e a c t i o n cycle h a v e a c t i v a t i o n energies in the 8-12 Kcal M-1 range. Thus, the p h o t o c h e m i c a l i n p u t of l h e first electron of t h e r e a c t i o n cycle r e m a i n s a v e r y fast process, even at l o w t e m p e r a t u r e . It shoutd be noted that the o x y - f e r r o c y t o c h r o me (Fe ~+. RH), w h i c h is stable for s e v e r a l hours, is r e a d i l y f o r m e d at subzero t e m p e r a t u r e s by the a d d i t i o n of 0 2 to the p h o t o r e d u c e d c y t o c h r o m e [19].
t
t Flash
683
T h e m i c r o s o m a l m e m b r a n e b o u n d or solubilized c y t o c h r o m e 'P450 c a n be p h o t o r e d u c e d in a sim i l a r m a n n e r by the t w o above d e s c r i b e d systems (MV. has even been s h o w n to be a n e c e s s a r y m e d i a t o r for the e l e c t r o n t r a n s f e r b e t w e e n dit h i o n i t e and f e r r i c c y t o c h r o m e [20]). W h e n oxygen is b u b b l e d t h r o u g h such a p h o t o c h e m i c a l l y r e d u c e d a n a e r o b i c solution at subzero t e m p e r a t u res, it consumes q u i c k l y the p h o t o r e d u c e d species. At t h e same time ~the oxygen b i n d s to Fe 2+ to f o r m an o x y - f e r r o c o m p o u n d , w h i c h is stable at - - 30°C and h i g h pa H (C. Bonfils, P. Maurel, P. Debey, to be published). In contrast, w h e n Na2S20 4 is used as a c h e m i c a l r e d u c t o r only, it reacts v e r y s l o w l y at subzero t e m p e r a t u r e s w i t h a d d e d oxygen and the r e m a i n i n g excess destroys r a p i d l y the oxyferro compound.
2.
PHOTODISSOCIATION
OF
THE
CARBOXY - F~BBO
COMPOUND.
As an a l t e r n a t i v e to the above, light can be used to i n t r o d u c e a r a p i d p e r t u r b a t i o n in a system p r e v i o u s l y in e q u i l i b r i u m . At low t e m p e r a t u r e s the subsequent r e t u r n to e q u i l i b r i u m is dras-
C. B o n f i l s a n d coll.
684
tieally slowed down, thus a l l o w i n g i n t e r m e d i a t e species to be t r a p p e d a n d characterized by r a p i d optical s c a n n i n g ,
c o m p o u n d at a n y t e m p e r a t u r e a n d CO c o n c e n t r a tion from 15 ~M to 80 ~M can be expressed as a m i x t u r e of two first o r d e r phases of n e a r l y iden% Photoreduction
k in sec -1
t
®
10 6
10C
kf
/
50
®
10 5
z.~O'
;.4
'
;.6
'
;.8 " 1~( 0K-1 )
0
50
~
50
-~o Temperature -'30 ~
FIG. 3. - Arrhenitts plot o f the fast (kt) arts slow (k~) rate consrants for recombination of CO to microsomal cytochrome P~so after flash photolysis. Solvent 50/50 (v/v) mixture of glycerol and 100 mM phosphate buffer pH 7.5. Temperature dependence of the photodissofiation yield 10 msec after the flash. (~ Solvent contains only 10 per cent (v/v) glycerol. (~ Solvent contains 50 per cent (v/v) glycerol.
a) Bacterial c y t o c h r o m e . R e c o m b i n a t i o n of the heme ligand w i t h CO after flash p h o t o d i s s o c i a t i o n of Fe2÷-CO b o n d has been studied over a wide range of subzero temperates. W i t h the bacterial cytochrome, the rec o m b i n a t i o n is first o r d e r m o n o p h a s i c at all temp e r a t u r e s studied, w i t h a rate constant of 3.5 104 M-1 see-1 at 12°C (i.e. very close to the value measured i n aqueous buffer at 4°C [21] 3.8 104 M-1 sec-1) a n d an activation energy of 8 -4- 0.4 Kcal M-1 in h y d r o - o r g a n i c medium. b) Microsomal c y t o c h r o m e . We have s i m i l a r l y studied the p h o t o d i s s o c i a t i o n of the carboxy-ferro complex of purified microsomal cytochrome P45o in glycerol-aqueous mixtures. Glycerol was chosen for its better p r o t e c t i n g effect on the m i c r o s o m a l cytochrome. The results are far more complex t h a n w i t h the bacterial cytochrome. The k i n e t i c s of the observed r e c o m b i n a t i o n after flash p h o t o d i s s o c i a l i o n of the carboxy-ferro
BIOCHIMIE, 1979, 61, n ° 5-6.
tical amplitude. The first phase is c.a. 6 to 8 times faster t h a n the last one, this ratio r e m a i n i n g constant w i t h temperature. The A r r h e n i u s plots of the two observed rate constants (k~ a n d k 0 are l i n e a r b e t w e e n + 20°C a n d - - 30°C w i t h an i d e n t i c a l activation energy of 11.5 -4- 1.5 K c a l / m o l e (figure 3). kf a n d k~ increase both p r o p o r t i o n a l l y to CO c o n c e n t r a t i o n up to 80 ~M. F i n a l l y w h e n the c o n c e n t r a t i o n of CO-free cytochrome is extrapolated to the time t = 0, only 70 per cent to 50 per cent p h o t o d i s s o c i a t i o n is f o u n d at + 20°C i n 50 per cent glycerol. This yield varies s o m e w h a t w i t h the p r e p a r a t i o n but r e m a i n s m u c h less t h a n 100 per cent i n p r e s e n c e of 50 per cent glycerol. It does not v a r y w i t h the CO c o n c e n t r a t i o n nor w i t h the flash power, n o r w i t h the p r e s e n c e of the detergent Renex (0.1 per cent or 0.5 per cent). W h e n t e m p e r a t u r e decreases the i n i t i a l p h o t o d i s s o c i a t i o n increases up to a m a x i m u m at c.a. - - 15°C -4- 3°C. I n presence of only 10 per cent glycerol, the r e c o m b i n a t i o n r e m a i n s b i p h a s i c but the i n i t i a l p h o t o d i s s o c i a t i o n yield is much higher (90 per
C y t o c h r o m e P~5o p h o t o c h e m i s t r y . cent to 100 per cent) a n d decreases slowly w h e n t e m p e r a t u r e decreases from 20°C to - - 8°C. Although the a m p l i t u d e a n d ratio of the two r e c o m b i n a t i o n phases a n d the i n i t i a l photodissoclarion yield v a r y q u a n t i t a t i v e l y somewhat ~vith the sample a n d its history, even ~vhen starting from the same stock solution, the basic features described above r e m a i n qualitatively u n c h a n g e d . F u r t h e r m o r e we checked in each case that the cytochrome was saturated w i t h CO and the soIution essentially free of P420.
685
boxy-ferro c o m p o u n d is too slow to allow spontaneous d i s p l a c e m e n t of CO b y 0 2 via : Fe 2÷ - -
kl CO ~- Fe 2* -[- CO k. 1
k2 Fe2+ + 02 ~ Fe 2 + _
(1)
02
(2)
k_2
However, 02 b i n d i n g is m u c h faster t h a n CO b i n d i n g . F u r t h e r m o r e , Fe2* __ 02 is only p o o r l y
3. FORMATION OF THE OXY-FERRO COMPOUND, Light p h o t o d i s s o c i a t i o n of m u l t i p l e ligand equil i b r i a alIows to form easily the oxy-ferro com-
i
0.05
t = .30°C
I
0.05
x
\ \ \
i
t
t
i
I
I
T
370
385
400
415
430
/=,45
i
460 L
in
nm
FIG. 4. - - Formation of the oxy-eomplex of bacterial cytochrome P~o after photodissociation of FeSc+o" RH in presence of 0~. Solvent 1:1 (v/v) mixture of 100 mM Na*/K+ phosphate buffer pH 7.3 and ethylene glycol (pare: 7.8 at 20°C) containing 280 ~tM camphor. Cytochrome P~5o, 6.16 ~tM ; CO. 20 ~tM ; t : 30°C ; wavelength interval 90 nm, speed 20 nm.sec-1. Reference wavelength 404 nm. (~) Fe~+'RH spectrum. (~) after 5 sec aeration with 0~. (~) after flash.
p o u n d from the carboxy-ferro c o m p o u n d . At - - 30°C the dissociation rate c o n s t a n t of the car-
BIOCHIMIE, 1979, 61, n ° 5-6.
i 380
i
i 400
i 420
i 440
\
i
t
k
;~ I n n m
Fro. 5 . - Spectral intermediates of bacterial cytochrome P~so at --20°C, (~) after flash photoreduction by FMNI-Lj (Fe~+RH), (~) after 02 bubbling (Fe~+'RH), (~) and (~ after several flashes on the same oxygenated solution. Solvent as in figure 2 with 5 mM EDTA, 300 ttM camphor, 6 ttM cytochrome (final concentrations). Dual mode spectral scan. Reference wavelength 404 nm.
photodissociable ; a c c o r d i n g l y flash photodissoclarion ~t - - 30°C of Fe 2÷ - - CO in the presence of 20 ~M CO and 100 ~M 02 (figure 4) results in the f o r m a t i o n of 100 p e r cent F e 2÷ - - 02 w i t h i n 10 msec. It is n o t e w o r t h y that at - - 30°C, Fe - R H is stable t o w a r d s a u t o x i d a t i o n (1/2 t ---- 8 hours) but not completely t o w a r d s l i g a n d exchange (slow d i s p l a c e m e n t of 02 b y CO).
C. BonfiIs and coll.
686 4. I N T E R A C T I O N
OF
THE
OXY-FERRO
CYTOCHROME
W I T H REI)UCING RADICALS.
A solution of the oxy-forro adduct p r e p a r e d in presence of a photoactive system (FMN or AO % l~fV) a n d stabilized at subzero temperatures, can be i r r a d i a t e d to p r o d u c e r e d u c i n g radicals. As s h o w n b y figure 5, successive flashes at - - 3 0 ° C on a Fe 2. - - 0 2 solution c o n t a i n i n g FMN p r o d u c e a progressive shift of the Sorer m a x i m u m from 418 (Fe~+ . RH) to 410 n m (Fe 2+ . RH). The n u m b e r of flashes (i.e. the total d u r a t i o n of illumin.ation) necessary for complete t r a n s f o r m a t i o n depends only on the oxygen c o n c e n t r a t i o n , all other conditions b e i n g identical. This clearly shows that the ouly effect of p h o t o r e d u c e d radicals is to consume oxygen progressively a n d shift the equilikl b r i u m Fe 2+. R,tI -t- 0~. ~ F e ~ . RH t o w a r d s re-
k.1 duction. After complete t r a n s f o r m a t i o n , a second oxygen b u b b l i n g restores the 418 n m b a n d . I d e n t i c a l resutts were o b t a i n e d w i t h the AO -F MV system. Thus n e i t h e r FMNH., nor FMNH 2 or MV- are able to t r a n s f e r the second electron to the oxy-ferro c o m p o u n d .
Discussion. P h o t o c h e m i c a l systems are very useful for studies on the first electron r e d u c t i o n of c y t o c h r o m e P450, since they p r o v i d e fast electron i n p u t even at low temperatures. It was quite s u r p r i s i n g however to observe that electrons p r o d u c e d in this m a n n e r were u n a b l e to react w i t h the oxygenated c o m p o u n d . I n the n a t u r a l h y d r o x y l a t i o n cycle addition of the second electron results in oxygen activation. These observations contrast also with the h i g h reactivity of the same r e d u c i n g agents w i t h formal analogs of oxygenated eytoehrome P45o, such as c o m p o u n d III of peroxydases [22]. These data emphasize the u n i q u e c h a r a c t e r of c y t o c h r o m e P45o a n d the definite r e q u i r e m e n t of a specific electron donating-effector p r o t e i n for the second electron r e d u c t i o n of the oxygenated complex [13]. Although not directly related to the enzymatic cycle, the k i n e t i c s and t h e r m o d y n a m i c s of carb o n m o n o x i d e b i n d i n g to the ferrous i r o n can be used to characterize the heme center. F o r the bacterial cytochrome, lhe r e c o m b i n a t i o n after flash photolysis of Fe2÷_ CO follows a simple first order r e a c t i o n in m i x e d solvent at a n y tempera-
BIOCHIMIE, 1979,
61, n ° 5-6.
ture above its freezing point, and provides a test of the i n o c u i t y of the o r g a n i c solvent. The u n i q u e features observed by flash photolysis of the purified nlierosomal cytochrome P450 are w o r t h to m e n t i o n a n d discuss. Similar results were already o b t a i n e d w i t h ~ h o l e microsomes or c r u d e p r e p a r a t i o n s of solubilized e y t o e h r o m e [6]. The two phases could h a r d l y r e p r e s e n t two pop u l a t i o n s of cytoehrome P4no since the preparation is electrophoreticaliy pure and, on the other h a n d , the h i g h l y heterogeneous m i e r o s o m a l population exhibits the same p r o p o r t i o n of fast and slow r e a c t i n g material [6]. F o r the same reason, they c a n n o t be linked to different p h y s i c a l (memb r a n e b o u n d or soluble) or a g g r e g a t i o n state, since Renex has no substantial effect. They could r a t h e r rely on different possible c o n f o r m a t i o n s of the molecule, r e l a x i n g on the 10 .2 sec to I sec time scale i n the -J- 20 ° - - 30°C t e m p e r a t u r e range. The i n c o m p l e t e a n d t e m p e r a t u r e - d e p e n d e n t (( i n i t i a l >> p h o t o d i s s o c i a t i o n c a n n o t he due to a low q u a n t u m yield since 100 per cent photodissociation is obtained w i t h 10 per cent glycerol. It r a t h e r i n d i c a t e s a t h i r d r a p i d phase o c c u r r i n g dur i n g the dead time (10 msec) of the method, similar to the process I ( r e c o m b i n a t i o n i n the pocket w i t h a cage effect) a l r e a d y observed by Austin et al. [10, 23] w i t h other soluble h e m o p r o l e i n s (myoglobin, hemoglobin, bacterial c y t o c h r o m e P450) at m u c h lower t e m p e r a t u r e s ( ( 2 0 0 ° K ) in frozen or glassy state. T h e i r photolysis experiments extended to the n a n o - s e c o n d range and cryogenic t e m p e r a t u r e s allowed to resolve the rec o m b i n a t i o n of a gazeous ligand in various elemen.tary processes a n d can serve as a basis for i n t e r p r e t i n g our present results on a more limited time and t e m p e r a t u r e scale. P a r t i c u l a r l y the glycerol a n d t e m p e r a t u r e effect on the yield could be thought out as a c o m p e t i t i o n between microviseosity a n d t e m p e r a t u r e effect on rate constants. The fact that two rate constants are observable at room t e m p e r a t u r e seems to i n d i c a t e that the mol e c u l a r r e l a x a t i o n b e t w e e n various c o n f o r m a t i o n s are very slow as c o m p a r e d w i t h other hemoproreins [10]. It w o u l d be of interest to evaluate the relevance of these p h y s i c o - e h e m i e a l proterties to the u n u s u a l properties of the liver h y d r o x y l a s e as c o m p a r e d to other c y t o c h r o m e P450 systems, i.e. its heterogeneity [24], a n d b r o a d specificity [25]. F r o m a more p r a c t i c a l p o i n t of view, c o m b i n a tion of high activation a n d cooling-heating cycles m a y overcome one of the major draw backs of the subzero t e m p e r a t u r e method, i.e. the high v i s e o -
Cytochrome P ~5o photochemistry. sity which sometimes prevents a proper mixing of r e a c t a n t s . T h e v a r i o u s p o s s i b i l i t i e s of t h i s m e t h o d are n o w b e i n g tested on other sties of /he c y t o c h r o m e P45o c y c l e a n d o t h e r m u l t i - c o m p o nents electron transport chains.
Acknowledgements. This w o r k w a s s u p p o r t e d by grants f r o m the l n s t i t u t N a t i o n a l de la Santd et de la Recherche Mddicale (U-128), f r o m the C.N.R.S., f r o m the D.G.R.S.T. (contrat n ° 7870332) and f r o m the F o n d a t i o n p o u r la Recherche M~dicale F r a n f a i s e .
REFERENCES. 1. Douzou P. (1977) Adv. in E n z y m o l . , 45, 157-272. 2. Douzou, P. (1977) , Acad. Press, London. 3. Cox, R. P. (1978) Biochem. Bey., 6, 689-697. 4. Hut Bon Hoa, G. & Douzou, P. (1972) Anal. Biochem., 51, 127-136. 5. Ballou, D. (1971) Ph. D. Thesis, University of Michigan, Ann Arbor, Michigan, U.S.A. 6. Debey, P., Balny, C. ~ Douzou, P. (1973) F E B S Letters. 35, 86-90. 7. Chance, B., Graham, N. ,~ Legallais~ V. (1975) Anal. Biochem., 67, 552-579. 8. Douzou, P., Sireix, R. ~ Travers, F. (1970) Proc. Natl. Acad. Sci. U.S.A., 66, 787-792. 9. Chance, B., Saronio, C. ~ Leigh, J. S. Jr. (1975) Proc. Natl. Acad. Sci. U.S.A., 72, 1635-1640.
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10. Austin, R. H., Beeson_ K. W., Eisenstein, L., F r a u e n felder, H. ,~ Gunsalus, I. C. (1975) B i o c h e m i s t r y , 14, 5355-5373. 11. Cox, R. (1975) B i o e h i m . B i o p h y s . Aeta, 387, 588-598. 12. Peterson, J. A., Ishimura, Y., Baron, J. ~ Estabrook, R. W. (1973) in
