Volume 85, number 1
FUNCTIONAL
FEBS LETTERS
STUDIES
ON CROSSL[NKED
BOVINE
January 1978
CY'[OCHROtVfE
c OXIDASE
Celia B O N A V E N T U R A and .~oseph B O N A V E N T U R A Dh~ke University Alarine Laboratory, Beaufort, North Carolina 28516, USA Maufizio BRUNORIr Centro di Bioiogia 3lolec6laire, Istituto di Chimica Biologica, Citta Universitaria, 00185 Rome, Italy and Michael W~LSON Department o f Chemi~stry, University o f Essex, [¢ivenhoe Park, Colchester, Essex, C04 3SQ, England Received
! . Ir~troduetion C y t e c h r o m e oxidase (EC 1.9.3.1 .) acts as the terminal electron z c c e p t o r in the m / t o c h o n d r i a l respir a t o r y chain and catalyzes the r e d u c t i o n o f m o l e c u l a r e x y g e n to water. It is c o m p r i s e d o f f o u r r e d o x sites, t w o heine a ~roups associated w i t h the c y t o c h r o m e s a and aa and t w o c o p p e r a t o m s distinguishable on b o t h functional and spectral grounds (reviews [1,2] ). It is k n o w n f r o m SDS gei electrophoresis t h a t c y t o c h r o m e oxidases consist o f at least six and poss;_bly eight p o l y p e p t i d e chains [3 --5 ~.~T:he relationships ~etween the functional properties o f the r e d o x sites and the p o l y p e p t i d e subuoAt structure o f the e n z y m e are, however, largely u n e x p l o r e d . R e c e - a t f u n c t i o n a ! studies on isolated c y t o c h r o m e oxidase have suggested that the oxidized e n z y m e , as p r e p a r e d , is o f s e c o n d a r y cata!yt,_'c i m p o r t a n c e b u t rather acts as a p o o l o f 'resting' e n z y m e f r o m which an activated species, t e r m e d 'pulsed' oxidase, m a y be generated in response to a flux o f electrons [ 6 ] . This activatiGn process takes place u p o n the e n z y m e c o m plex uncergoing o n e cycle o f full reduction and reaction w i t h molecular o x y g e n and alse goes part w a y t o explain k n o w n discrepancies b e t w e e n the e n z y m e - t u r n o v e r n u m b e r as measured b y transient and steady-state m e t h o d s . 30
7 October
1977
Kn this p a p e r we r e p o r t findings t h a t indicate t h a t activation o f the oxidase can be accompl/shed n o t o n l y for the isolated e n z y m e b u t also w h e n it is i m b e d d e d in the m e m b r a n e o f the s u b m i t o c h o n d r / a l particles thus indicating a possible in vivo i m p o r t a n c e for this activation. Also to investigate the role p l a y e d b y c o n f o r m a t i o n a I rearrangement b e t w e e n subunits in this activation process we have u n d e r t a k e n crosslinldng e x p e r i m e n t s with purified e n z y m e in an a t t e m p t to impair the c o n f o r m a t i o n a l flexibility o f the oxidase assembly.
2. MateriMs a n d m e t h o d s C y t o c h r o m e ox/dase and Keiiin-Hartree particles were prepared as described [ 7 ] . CrossiinkJng was carried o u t b y the m e t h o d [8] using d i m e t h y l subefim/date. Solutions, 25 geM, o f either fully oxidized or dithiordte reduced c y t o c h r o m e oxidase were reacted at r o o m t e m p e r a t u r e w i t h d i m e t h y l suberimidate for 3 h. The reaction was s t o p p e d b y the a d d i t ' o n o f a m m o n i u m chlor/de to a final c o n c e n t r a t i o n o f 0.1 M a n d the reaction m i x t u r e was t h e n dialyzed against 0.1 M p o t a s s : u m p h o s p h a t e buffer, p H 7.4, containing 1% T w e e n 80 overnight. Atl samples were in the fully oxidized state a f t e r Elsevier/North-Hoitand A~'omedicalPress
Volume 85, number 1
FEBS LETTERS
dialysis, as j u d g e d b y t h e i r s p e c t r a . As a c o n t r o l a 25/A~i s o l u t i o n o f o x i d i z e d o x i d a s e was t r e a t e d i n a n i d e n t i c a l m a n n e r e x c e p t t h a t s u b e f i m i d a t e was omffted from the reaction mixture. The c o n c e n t r a t i o n o f c y t o c h r o m e oxidase in terms o f h e r n e was d e t e r m i n e d u s i n g c = 21 0 0 9 M - ~c m - ~ at 6 0 5 n m f o r t h e f u l l y r e d u c e d e n z y m e [7]. T h e oxddase c o n c e n t r a t i o n s are, h o w e v e r , e x p r e s s e d i n t e r m s o f a tWO h e i n e (aa3) c o m p l e x . S D S gel e l e c t r o p h o r e s i s was
January 1978 t i m e { rafltisec ] 1130
2~30
300
~00
100
~0
6O
Xn
20
c
p e r l 0 r m e d according to [ ! 2 ] . I
t
t
1
2
3
I
i "
4
time {see }
:3. g.esul~s a n d d l s c u s s [ o n
3.1. SDS gels F i g u r e t s h o w s SDS gels o f c y t o c h r o m e o x i d a s e w h i c h have b e e n c r o s s l i n k e d i n e i t h e r t h e f u l l y o x i d i z e d o r the f u l l y r e d u c e d s t a t e a n d c o m p a r e s t h e s e w i t h uncrossHnked enzyme. The uncrosslinked enzyme exhibits a multibanded pattern in agreement with [3--5 ] a n d i n d i c a t e s t h e p r e s e n c e o f e i g h t p o l y p e p t ~ d e chahas w i t h a p p r o x , tool. w t range 5 ! 0 0 0 - - 1 6 0 0 0 , together with some further higher molecular weight c o m p o n e n t s . TbAs p a t t e r n m a y b e c o n t r a s t e d w i t h t h a t of the crosslinked enzyme. F~ ~
[t is a p p a r e n t t h a t s u b e r i m i d a t e has l i n k e d t o g e t h e r s o m e o f the p o l y p e p t i d e c h a i n s a n d o n l y t w o l o w e r m o l e c u l a r weiojat c o m p o n e n t s ( a p p r o x . tool. w t 23 0 0 0 , 15 0 0 0 ) are n o w e v i d e n t , t h e r e m a i n d e r o f the e n z y m e failing t o e n t e r t h e gel.
3.2. Functional studies
L.A
Control
Fig.2. Electron transfer b e t v e e n horse ferrocytochrome c and bovine cytochrome oxidase in the uncrosslinked and cross!inked condition. Cytochrome oxidase concentration 1.3 I~M; ferrocytochrome c 2 #M. C, X O and X R refer to the control, oxidase crosslinked in the oxidised condition and reduced condition, respectively. The upper time scale refers to the top three traces and the lower scale to the bottom three traces. Monitoring wavelength 550 rim. Potassium phosphate buffer 0. l M, pH 7.4, containing 1% Tween 80. Temp. 20°(2.
M XO
XR
Fig. 1. Polyacrylamide ge!-SDS electrophoresis performed according to the method [ 12] and Ln the presence of the proteLrtase hahibitor. The major band app. t o o l wts axe: Control, 50 700, 44 700, 35 500, 30 200, 27 900, 22 400, !9 500 and 16 200; X O, 24 000 and 14 500: X R, 22 900 and 15 800.
F i g u r e 2 s h o w s typical progress curves o f the e l e c t r o n t r a n s f e r r e a c t i o n b e t w e e n ferrocytochrorne c a n d c y t o c h r o m e oxidase at l o w p r o t e i n c o n c e n t r a t i o n s _ [ n a g r e e m e n t w i t h [9] we observe a fast process ( s e c o n d - o r d e r rate a p p r o x . 3 X 106 M -a s - * ) corres p o n d i n g t o the o x i d a t i o n o f c y t o c h r o m e e a n d a s i m u l t a n e o u s r e d ~ c t i o n o f c y t o c h r o m e a. W h e n m o n i t o r e d at a w a v e l e n g t h o f 5 5 0 n m this r e a c t i o n is followed b y a slower oxidation o f c y t o c h r o m e c which b y r e f e r e n c e to t h e photochenfical e x p e r i m e n t s [tff] a p p e a r s t o b e r a t e - l i m i t e d b y t h e i n t e n m l t r a n s f e r of" e l e c t r o n s f r o m c y t o e h r o m e a t o a3. It is clear t h a t the c r o s s l i n k e d e n z y m e b e h a v e s identic'Mly t o t h e u n t r e a t e d e n z y m e w i t h regard t o the rate a t w h i c h e l e c t r o n s e n t e r the e n z y m e f r o m c y t o c h r o m e c. This s i m i l a r i t y i n the f u n c t i o n a l p r o p e r t i e s o f t h e cro_~slinked a n d u n c r o s s U n k e d o x i d a s e is also seen i n t h e i r r e a c t i o n s 31
Volume 85, number I
FEBS LETTERS
January 1978 Table 1
w i t h carbon m o n o x i d e . Table 1 c o m p a r e s the rates o f c o m b i n a t i o n o f C O ~ S t h d i t h / o n i t e r e d u c e d enz3Tme as m e a s u r e d b y s t o p p e d - f l o w a n d p h o t o l y t i e m e t h o d s .
Reaction o f reduced uncross~ke.c~ ~nd c r o s ~ e d w / ~ carbon m o n o ~ d e
T h e s e rates are t h e s ~ m e in all cases a n d are in c l o s e a g r e e m e n t w i t h the vzlues for the c o m b i n a t i o n rate c o n s t ~ n : o f C O ,.¢/~d~red~=_ced c y t o c h r o m e o_~d~se [ 1 1 ] T h e fast e l e c : r o n e n t r y i n t o t h e o x i d a s e m o l e c u l e a n d t h e u m m p a / r e d l i g a n d b i n d g n g p r o p e r t i e s o f crossl i n k e d e:,~zyme suggests t h a t t h e c y t o c h r o m e c / n t e r a c t i o n site, c y t o c h r o m e a , a n d t h e a c c e s s i b i l i t y a n d b i n d i n g p r o p e r t i e s o f zhe h e i n e a s s o c i a t e d w i t h c y t o c h r o m e 2s are u n a f f e c t e d b y the c r o s s l i n l d n g o f t h e c o n s t i t u e n t p o l y p e p t i d e chains o f the o×idase comple×. In fig .3 w e r e p o r t p r o g r e s s c u r v e s m o n i t o r e d a t
Protein
Protein (~M)
CO (t~M)
rate (a e) (s -I )
Method
Control Control
4A 4.4 5.8 5.8
250 250 250 250
15.00 13.53 15.37 t4.5
5.5 5.5
258
15.56
F~ow Flash Flow Flash Row
250
15.08
Flash
X0 XR XR
Pota~ium phosphate, 0.1 M, pH 7.4, 1% Tween-80. Temp. 20°C
1.0
1-0
0-5
0-5
/~,At
Z~At
AAo
Z~Ao R
0-1
0-1
0
~ P
I
I
1
I
2 time
0
0-5
0-5
ZXAt
/%At
AAo
AAo
13
04
P
R
1
5
10 time
t sec }
10 time ( sec ]
1-0
!
5
(sec)
1-0
0-1
oxid~
I 0
R
5
10 t i m e { ~ec )
Fig.3. Oxidation o f horse ferrocytochrome c by membrane bound, isolated and crossl/~ked bov/ae cytochrome ox/dase. (a) Keil/n-Hartzee paxticles, oxidas~ conc~.'ntration 2.1. u~{, cytochrome c. 10 gM. (b) [sol~ted o.~dax~ 1.3 ~M, cytocI'~ome c, 6_25 gM, re) Isolated oxlda.~ crosslinked while in the oxid/sed state 1.3 ~zM, cytochromc c, 6.25 ~M. (d) isolated oxidas¢ c~oss~nked while in the reduced state 1.3 _~M, cytochrome c, 6.25 gM. R and P denote experiments with ~'1c "gcstmg" or "pu]scd" e_~.:yme (~.'e text) respect/rely. Experime:ats were performed in 0.1 M potassium phosphate buffex, pH 7.4, which, for those expetLments using the isola'zed enzyme, also contained 1% Tween 80. Oxyger~ concentration 135 #M, sodium aseorhate I raM. Monitoring wavelength 55Onto. Temp. 20~C. 32
V o l u m e 85, n u m b e r t
FEBS LETTERS
550 n m for hhe reaction b e t w e e n f e r r o c y t o c h r o m e c and either isolated c y t o c h r o m e orddase, crosslinked and uncrosslinked, or Keflin-Hartree particles. The concentrations are chosen so that each molecule o f c y t o c h r o m e oxidase will t u r n over a p p r o x i m a t e l y once on average, that ts the f e r r o c y t o c h r o m e c concentration ~s a p p r o x i m a t e l y equal t o the concentration o f redox sites fn the oxAdase or 4-fold greater than the total oxidase c o m p l e x concentration. This reaction has been studied in two ways. Firstly, b y mixing an aerobic solution o f oxidized "resting" c y t o c h r o m e oxidase with ascorbate-reduced c y t o c h r o m e c and secondly, b y reacting a mL~:ture o f ascorbate-reduced c y t o c h r o m e c and c y t o c h r o m e oxidase with an oxygen-containing buffer. In the first
m e t h o d we observe the oxidation o f f e r r o c y t o c h r o m e c b y c y t o c h r o m e oxidase which has been in the oxidized or "resting" state for a long period o f time while in the second m e t h o d we see the action o f freslflyoxidized oxidase or "pulsed" oxidase, which is p r o d u c e d within the deadtime o f the stopped-flow apparatus b y the oxidation o f oxldase b y molecular oxygen. As reported t'or uncrosslinked oxid.ase [6~ the two m e t h o d s show clearly different rates as ~lustrated in fig.3b and table 2, indicating that the rate o f internal electron transfer within the pulsed e n z y m e is approx. 3-fold greater than in the resting e n z y m e . The rate o f reaction o f the crossfinked e n z y m e , however, is the same b y b o t h m e t h o d s and the same as the rate exhibited b y the uncrosslinked, resting e n z y m e (see table 2). We m a y conclude from this experiment that whereas the crosslinked e n z y m e is able t o oxidize Table 2 Rate of the slow phase of oxidation of ferrocytochrome c
J a n u a r y 1978
f e r r o c y t o c h r o m e c as rapidly as uncrosslinked resting e n z y m e it is unable to b e c o m e activated and display the enhanced rates that are characteristic o f the un,:reated e n z y m e . The differences between the crosslinked and uncrosslinked e n z y m e s reside in the fact that the subunits are linked together in the f o r m e r case and, presumably, this interferes with the c o n f o r m a t i o n a ! flexAbflity o f the molecules. The activation process w o u l d therefore seem to rely on the oxidase molecule entering in its activated state a c o n f o r m a t i o n different from either the fully-oxidized resting e n z y m e o~- the tully-reduced e n z y m e . This activated state corresponds to the c o n d i t i o n o f the e n z y m e when it is turning over, as a cycle o f reduction and oxidation in a steadystate experiment should produce the activated e n z y m e in an analogous fashion to the way described above. [n order to ascertain whether the activation process is an artifact generated b y the isolation procedure we have c o n d u c t e d some experiments on Keflin-Hartree particles. In fig.3 the resting and pulsed particles are c o m p a r e d in their rate o f oxidation o f f e r r o c y t o c h r o m e c. It is clear that the activation process occurs leading to a 3-fold e n h a n c e m e n t o f the rate. The fact that this e n h a n c e m e n t occurs in the m e m b r a n e - b o u n d e n z y m e suggests that it is physiologically relevant and that the activation process m a y have an i m p o r t a n t role in the c o n t r o l o f the respiratory chain as discussed [~]. We also see (table 2, fig.3) that under comparabb~ ~ondb tions the ~x,embra~e-bound oxidase oxidises ~erroc y t o c h r o m e c consxderably faster ( ~ 5-fold) than the isolated enzyme. This is in agreement with the k n o w n activation o f c y t o c h r o m e oxidase b y !ipids [ 13 ].
Acknowledgements
"Resting"
"Pulsed"
Oxidase in
1.25
3.95
This w o r k was supported b y National Science F o u n d a t i o n G r a n t PCM 75436451. M. T. ~ l s o n gratefully acknowledges grants from Duke University, Biomedical Reseazch S u p p o r t Grant from the National Institute o f Health. M. Brunori expresses his thanks to The National Research Council (CNR) o f Italy for travel support.
p~ur~icles Cont-rc& XO
0.26 0.25
O. 74 0.28
References
XR
0.33
0.24
by m e m b r a n e - b o u n d , isolated and crosslinked bovine c y t o c h r o m e oxddase R a t e (a-I) Protein
Values taken f r o m p l o t s ha fig.3
[ ! ] Lemberg, M. R. and Barret, L (.[973) in: Cytochromes, Academic Pre~s. 33
Volume 85, number !
FEBSLETTERS
[:!] Nit:hot,ls, P. and Chance, B. (1974) in: M o [ e c u ~ ~V~cch,~t/sr~s o f OXygen A c t i v a t i o n (Hayaisiu, O. dd) pp. 4 7 9 - - 5 3 4 , A c a d e m i c Pxess, New York. [3] Ey~on, G. D., Rogezs, C. C., Shatz, G. a n d Racker, E. ( 1 9 7 5 ) J. Biol. C h e m . 250, 8 5 9 8 8 6 0 3 . [~:] Briggs, M., K a m p , P. F., R o b i n s o n , N. C. a n d Capaldi, R_ C_ ( 1 9 7 5 ) B~;ochem/stry 14. 5 1 2 3 - - 5 1 2 ~ . [5] B u : h e r , $. R. a n d PennmU, R. ( 1 9 7 5 ) F E B S Lett. 60, I80--184.
[6] AntoninL E., Bzunori, M., Colosimo, A., Greenwood, C. a n d Wilson, M. T. (1977) Pzoc. Natl. Acad. Sci. USA 74. 3 1 2 8 - - 3 1 3 2 . [7] Y o n e t a n i , T_ ( 1 9 6 0 ) i_ BioL Chem. 2 3 5 , 8 4 5 - - 8 5 2 .
34
tanu~y
1978
[8] Davies, G. E. ~nd Stark, G. R. ( 1 9 7 0 ) Pzoc. l~atl. Aead. Sci. U:~A 66, 651--6S6. [9] W~son, M. T., G r e e n w o o d , C., Brunod, M. and A n t o n h t i ,
E. (1975) Biochem. L 147, 145-153. [10] G i b s o n , Q. H., G r e e n w o o d , C., W h a r t o n , D. C. a n d P , ~ n e r , G. ( 1 9 6 5 ) J . Biol. C h e m . 2 4 0 , g 8 8 894. [ 11 ] Gibsor:, Q. H. a n d G r e e n w o o d , C. ( 1 9 6 3 ) B i o c h e m . 3. 86, 5 4 [ 5 5 4 . [12] Weber, K. a n d O s b o r n e , M. ( 1 9 6 9 ) J. Biol. C h e m . 244, 4406-44t2. [13] Wharton, D. C. a n d Grfffiths, D. E. ( 1 9 6 2 ) Arch. Bfochem. Bgophys. 9 6 , 1 0 3 ~_14.
FUNCTIONAL
FEBS LETTERS
STUDIES
ON CROSSL[NKED
BOVINE
January 1978
CY'[OCHROtVfE
c OXIDASE
Celia B O N A V E N T U R A and .~oseph B O N A V E N T U R A Dh~ke University Alarine Laboratory, Beaufort, North Carolina 28516, USA Maufizio BRUNORIr Centro di Bioiogia 3lolec6laire, Istituto di Chimica Biologica, Citta Universitaria, 00185 Rome, Italy and Michael W~LSON Department o f Chemi~stry, University o f Essex, [¢ivenhoe Park, Colchester, Essex, C04 3SQ, England Received
! . Ir~troduetion C y t e c h r o m e oxidase (EC 1.9.3.1 .) acts as the terminal electron z c c e p t o r in the m / t o c h o n d r i a l respir a t o r y chain and catalyzes the r e d u c t i o n o f m o l e c u l a r e x y g e n to water. It is c o m p r i s e d o f f o u r r e d o x sites, t w o heine a ~roups associated w i t h the c y t o c h r o m e s a and aa and t w o c o p p e r a t o m s distinguishable on b o t h functional and spectral grounds (reviews [1,2] ). It is k n o w n f r o m SDS gei electrophoresis t h a t c y t o c h r o m e oxidases consist o f at least six and poss;_bly eight p o l y p e p t i d e chains [3 --5 ~.~T:he relationships ~etween the functional properties o f the r e d o x sites and the p o l y p e p t i d e subuoAt structure o f the e n z y m e are, however, largely u n e x p l o r e d . R e c e - a t f u n c t i o n a ! studies on isolated c y t o c h r o m e oxidase have suggested that the oxidized e n z y m e , as p r e p a r e d , is o f s e c o n d a r y cata!yt,_'c i m p o r t a n c e b u t rather acts as a p o o l o f 'resting' e n z y m e f r o m which an activated species, t e r m e d 'pulsed' oxidase, m a y be generated in response to a flux o f electrons [ 6 ] . This activatiGn process takes place u p o n the e n z y m e c o m plex uncergoing o n e cycle o f full reduction and reaction w i t h molecular o x y g e n and alse goes part w a y t o explain k n o w n discrepancies b e t w e e n the e n z y m e - t u r n o v e r n u m b e r as measured b y transient and steady-state m e t h o d s . 30
7 October
1977
Kn this p a p e r we r e p o r t findings t h a t indicate t h a t activation o f the oxidase can be accompl/shed n o t o n l y for the isolated e n z y m e b u t also w h e n it is i m b e d d e d in the m e m b r a n e o f the s u b m i t o c h o n d r / a l particles thus indicating a possible in vivo i m p o r t a n c e for this activation. Also to investigate the role p l a y e d b y c o n f o r m a t i o n a I rearrangement b e t w e e n subunits in this activation process we have u n d e r t a k e n crosslinldng e x p e r i m e n t s with purified e n z y m e in an a t t e m p t to impair the c o n f o r m a t i o n a l flexibility o f the oxidase assembly.
2. MateriMs a n d m e t h o d s C y t o c h r o m e ox/dase and Keiiin-Hartree particles were prepared as described [ 7 ] . CrossiinkJng was carried o u t b y the m e t h o d [8] using d i m e t h y l subefim/date. Solutions, 25 geM, o f either fully oxidized or dithiordte reduced c y t o c h r o m e oxidase were reacted at r o o m t e m p e r a t u r e w i t h d i m e t h y l suberimidate for 3 h. The reaction was s t o p p e d b y the a d d i t ' o n o f a m m o n i u m chlor/de to a final c o n c e n t r a t i o n o f 0.1 M a n d the reaction m i x t u r e was t h e n dialyzed against 0.1 M p o t a s s : u m p h o s p h a t e buffer, p H 7.4, containing 1% T w e e n 80 overnight. Atl samples were in the fully oxidized state a f t e r Elsevier/North-Hoitand A~'omedicalPress
Volume 85, number 1
FEBS LETTERS
dialysis, as j u d g e d b y t h e i r s p e c t r a . As a c o n t r o l a 25/A~i s o l u t i o n o f o x i d i z e d o x i d a s e was t r e a t e d i n a n i d e n t i c a l m a n n e r e x c e p t t h a t s u b e f i m i d a t e was omffted from the reaction mixture. The c o n c e n t r a t i o n o f c y t o c h r o m e oxidase in terms o f h e r n e was d e t e r m i n e d u s i n g c = 21 0 0 9 M - ~c m - ~ at 6 0 5 n m f o r t h e f u l l y r e d u c e d e n z y m e [7]. T h e oxddase c o n c e n t r a t i o n s are, h o w e v e r , e x p r e s s e d i n t e r m s o f a tWO h e i n e (aa3) c o m p l e x . S D S gel e l e c t r o p h o r e s i s was
January 1978 t i m e { rafltisec ] 1130
2~30
300
~00
100
~0
6O
Xn
20
c
p e r l 0 r m e d according to [ ! 2 ] . I
t
t
1
2
3
I
i "
4
time {see }
:3. g.esul~s a n d d l s c u s s [ o n
3.1. SDS gels F i g u r e t s h o w s SDS gels o f c y t o c h r o m e o x i d a s e w h i c h have b e e n c r o s s l i n k e d i n e i t h e r t h e f u l l y o x i d i z e d o r the f u l l y r e d u c e d s t a t e a n d c o m p a r e s t h e s e w i t h uncrossHnked enzyme. The uncrosslinked enzyme exhibits a multibanded pattern in agreement with [3--5 ] a n d i n d i c a t e s t h e p r e s e n c e o f e i g h t p o l y p e p t ~ d e chahas w i t h a p p r o x , tool. w t range 5 ! 0 0 0 - - 1 6 0 0 0 , together with some further higher molecular weight c o m p o n e n t s . TbAs p a t t e r n m a y b e c o n t r a s t e d w i t h t h a t of the crosslinked enzyme. F~ ~
[t is a p p a r e n t t h a t s u b e r i m i d a t e has l i n k e d t o g e t h e r s o m e o f the p o l y p e p t i d e c h a i n s a n d o n l y t w o l o w e r m o l e c u l a r weiojat c o m p o n e n t s ( a p p r o x . tool. w t 23 0 0 0 , 15 0 0 0 ) are n o w e v i d e n t , t h e r e m a i n d e r o f the e n z y m e failing t o e n t e r t h e gel.
3.2. Functional studies
L.A
Control
Fig.2. Electron transfer b e t v e e n horse ferrocytochrome c and bovine cytochrome oxidase in the uncrosslinked and cross!inked condition. Cytochrome oxidase concentration 1.3 I~M; ferrocytochrome c 2 #M. C, X O and X R refer to the control, oxidase crosslinked in the oxidised condition and reduced condition, respectively. The upper time scale refers to the top three traces and the lower scale to the bottom three traces. Monitoring wavelength 550 rim. Potassium phosphate buffer 0. l M, pH 7.4, containing 1% Tween 80. Temp. 20°(2.
M XO
XR
Fig. 1. Polyacrylamide ge!-SDS electrophoresis performed according to the method [ 12] and Ln the presence of the proteLrtase hahibitor. The major band app. t o o l wts axe: Control, 50 700, 44 700, 35 500, 30 200, 27 900, 22 400, !9 500 and 16 200; X O, 24 000 and 14 500: X R, 22 900 and 15 800.
F i g u r e 2 s h o w s typical progress curves o f the e l e c t r o n t r a n s f e r r e a c t i o n b e t w e e n ferrocytochrorne c a n d c y t o c h r o m e oxidase at l o w p r o t e i n c o n c e n t r a t i o n s _ [ n a g r e e m e n t w i t h [9] we observe a fast process ( s e c o n d - o r d e r rate a p p r o x . 3 X 106 M -a s - * ) corres p o n d i n g t o the o x i d a t i o n o f c y t o c h r o m e e a n d a s i m u l t a n e o u s r e d ~ c t i o n o f c y t o c h r o m e a. W h e n m o n i t o r e d at a w a v e l e n g t h o f 5 5 0 n m this r e a c t i o n is followed b y a slower oxidation o f c y t o c h r o m e c which b y r e f e r e n c e to t h e photochenfical e x p e r i m e n t s [tff] a p p e a r s t o b e r a t e - l i m i t e d b y t h e i n t e n m l t r a n s f e r of" e l e c t r o n s f r o m c y t o e h r o m e a t o a3. It is clear t h a t the c r o s s l i n k e d e n z y m e b e h a v e s identic'Mly t o t h e u n t r e a t e d e n z y m e w i t h regard t o the rate a t w h i c h e l e c t r o n s e n t e r the e n z y m e f r o m c y t o c h r o m e c. This s i m i l a r i t y i n the f u n c t i o n a l p r o p e r t i e s o f t h e cro_~slinked a n d u n c r o s s U n k e d o x i d a s e is also seen i n t h e i r r e a c t i o n s 31
Volume 85, number I
FEBS LETTERS
January 1978 Table 1
w i t h carbon m o n o x i d e . Table 1 c o m p a r e s the rates o f c o m b i n a t i o n o f C O ~ S t h d i t h / o n i t e r e d u c e d enz3Tme as m e a s u r e d b y s t o p p e d - f l o w a n d p h o t o l y t i e m e t h o d s .
Reaction o f reduced uncross~ke.c~ ~nd c r o s ~ e d w / ~ carbon m o n o ~ d e
T h e s e rates are t h e s ~ m e in all cases a n d are in c l o s e a g r e e m e n t w i t h the vzlues for the c o m b i n a t i o n rate c o n s t ~ n : o f C O ,.¢/~d~red~=_ced c y t o c h r o m e o_~d~se [ 1 1 ] T h e fast e l e c : r o n e n t r y i n t o t h e o x i d a s e m o l e c u l e a n d t h e u m m p a / r e d l i g a n d b i n d g n g p r o p e r t i e s o f crossl i n k e d e:,~zyme suggests t h a t t h e c y t o c h r o m e c / n t e r a c t i o n site, c y t o c h r o m e a , a n d t h e a c c e s s i b i l i t y a n d b i n d i n g p r o p e r t i e s o f zhe h e i n e a s s o c i a t e d w i t h c y t o c h r o m e 2s are u n a f f e c t e d b y the c r o s s l i n l d n g o f t h e c o n s t i t u e n t p o l y p e p t i d e chains o f the o×idase comple×. In fig .3 w e r e p o r t p r o g r e s s c u r v e s m o n i t o r e d a t
Protein
Protein (~M)
CO (t~M)
rate (a e) (s -I )
Method
Control Control
4A 4.4 5.8 5.8
250 250 250 250
15.00 13.53 15.37 t4.5
5.5 5.5
258
15.56
F~ow Flash Flow Flash Row
250
15.08
Flash
X0 XR XR
Pota~ium phosphate, 0.1 M, pH 7.4, 1% Tween-80. Temp. 20°C
1.0
1-0
0-5
0-5
/~,At
Z~At
AAo
Z~Ao R
0-1
0-1
0
~ P
I
I
1
I
2 time
0
0-5
0-5
ZXAt
/%At
AAo
AAo
13
04
P
R
1
5
10 time
t sec }
10 time ( sec ]
1-0
!
5
(sec)
1-0
0-1
oxid~
I 0
R
5
10 t i m e { ~ec )
Fig.3. Oxidation o f horse ferrocytochrome c by membrane bound, isolated and crossl/~ked bov/ae cytochrome ox/dase. (a) Keil/n-Hartzee paxticles, oxidas~ conc~.'ntration 2.1. u~{, cytochrome c. 10 gM. (b) [sol~ted o.~dax~ 1.3 ~M, cytocI'~ome c, 6_25 gM, re) Isolated oxlda.~ crosslinked while in the oxid/sed state 1.3 ~zM, cytochromc c, 6.25 ~M. (d) isolated oxidas¢ c~oss~nked while in the reduced state 1.3 _~M, cytochrome c, 6.25 gM. R and P denote experiments with ~'1c "gcstmg" or "pu]scd" e_~.:yme (~.'e text) respect/rely. Experime:ats were performed in 0.1 M potassium phosphate buffex, pH 7.4, which, for those expetLments using the isola'zed enzyme, also contained 1% Tween 80. Oxyger~ concentration 135 #M, sodium aseorhate I raM. Monitoring wavelength 55Onto. Temp. 20~C. 32
V o l u m e 85, n u m b e r t
FEBS LETTERS
550 n m for hhe reaction b e t w e e n f e r r o c y t o c h r o m e c and either isolated c y t o c h r o m e orddase, crosslinked and uncrosslinked, or Keflin-Hartree particles. The concentrations are chosen so that each molecule o f c y t o c h r o m e oxidase will t u r n over a p p r o x i m a t e l y once on average, that ts the f e r r o c y t o c h r o m e c concentration ~s a p p r o x i m a t e l y equal t o the concentration o f redox sites fn the oxAdase or 4-fold greater than the total oxidase c o m p l e x concentration. This reaction has been studied in two ways. Firstly, b y mixing an aerobic solution o f oxidized "resting" c y t o c h r o m e oxidase with ascorbate-reduced c y t o c h r o m e c and secondly, b y reacting a mL~:ture o f ascorbate-reduced c y t o c h r o m e c and c y t o c h r o m e oxidase with an oxygen-containing buffer. In the first
m e t h o d we observe the oxidation o f f e r r o c y t o c h r o m e c b y c y t o c h r o m e oxidase which has been in the oxidized or "resting" state for a long period o f time while in the second m e t h o d we see the action o f freslflyoxidized oxidase or "pulsed" oxidase, which is p r o d u c e d within the deadtime o f the stopped-flow apparatus b y the oxidation o f oxldase b y molecular oxygen. As reported t'or uncrosslinked oxid.ase [6~ the two m e t h o d s show clearly different rates as ~lustrated in fig.3b and table 2, indicating that the rate o f internal electron transfer within the pulsed e n z y m e is approx. 3-fold greater than in the resting e n z y m e . The rate o f reaction o f the crossfinked e n z y m e , however, is the same b y b o t h m e t h o d s and the same as the rate exhibited b y the uncrosslinked, resting e n z y m e (see table 2). We m a y conclude from this experiment that whereas the crosslinked e n z y m e is able t o oxidize Table 2 Rate of the slow phase of oxidation of ferrocytochrome c
J a n u a r y 1978
f e r r o c y t o c h r o m e c as rapidly as uncrosslinked resting e n z y m e it is unable to b e c o m e activated and display the enhanced rates that are characteristic o f the un,:reated e n z y m e . The differences between the crosslinked and uncrosslinked e n z y m e s reside in the fact that the subunits are linked together in the f o r m e r case and, presumably, this interferes with the c o n f o r m a t i o n a ! flexAbflity o f the molecules. The activation process w o u l d therefore seem to rely on the oxidase molecule entering in its activated state a c o n f o r m a t i o n different from either the fully-oxidized resting e n z y m e o~- the tully-reduced e n z y m e . This activated state corresponds to the c o n d i t i o n o f the e n z y m e when it is turning over, as a cycle o f reduction and oxidation in a steadystate experiment should produce the activated e n z y m e in an analogous fashion to the way described above. [n order to ascertain whether the activation process is an artifact generated b y the isolation procedure we have c o n d u c t e d some experiments on Keflin-Hartree particles. In fig.3 the resting and pulsed particles are c o m p a r e d in their rate o f oxidation o f f e r r o c y t o c h r o m e c. It is clear that the activation process occurs leading to a 3-fold e n h a n c e m e n t o f the rate. The fact that this e n h a n c e m e n t occurs in the m e m b r a n e - b o u n d e n z y m e suggests that it is physiologically relevant and that the activation process m a y have an i m p o r t a n t role in the c o n t r o l o f the respiratory chain as discussed [~]. We also see (table 2, fig.3) that under comparabb~ ~ondb tions the ~x,embra~e-bound oxidase oxidises ~erroc y t o c h r o m e c consxderably faster ( ~ 5-fold) than the isolated enzyme. This is in agreement with the k n o w n activation o f c y t o c h r o m e oxidase b y !ipids [ 13 ].
Acknowledgements
"Resting"
"Pulsed"
Oxidase in
1.25
3.95
This w o r k was supported b y National Science F o u n d a t i o n G r a n t PCM 75436451. M. T. ~ l s o n gratefully acknowledges grants from Duke University, Biomedical Reseazch S u p p o r t Grant from the National Institute o f Health. M. Brunori expresses his thanks to The National Research Council (CNR) o f Italy for travel support.
p~ur~icles Cont-rc& XO
0.26 0.25
O. 74 0.28
References
XR
0.33
0.24
by m e m b r a n e - b o u n d , isolated and crosslinked bovine c y t o c h r o m e oxddase R a t e (a-I) Protein
Values taken f r o m p l o t s ha fig.3
[ ! ] Lemberg, M. R. and Barret, L (.[973) in: Cytochromes, Academic Pre~s. 33
Volume 85, number !
FEBSLETTERS
[:!] Nit:hot,ls, P. and Chance, B. (1974) in: M o [ e c u ~ ~V~cch,~t/sr~s o f OXygen A c t i v a t i o n (Hayaisiu, O. dd) pp. 4 7 9 - - 5 3 4 , A c a d e m i c Pxess, New York. [3] Ey~on, G. D., Rogezs, C. C., Shatz, G. a n d Racker, E. ( 1 9 7 5 ) J. Biol. C h e m . 250, 8 5 9 8 8 6 0 3 . [~:] Briggs, M., K a m p , P. F., R o b i n s o n , N. C. a n d Capaldi, R_ C_ ( 1 9 7 5 ) B~;ochem/stry 14. 5 1 2 3 - - 5 1 2 ~ . [5] B u : h e r , $. R. a n d PennmU, R. ( 1 9 7 5 ) F E B S Lett. 60, I80--184.
[6] AntoninL E., Bzunori, M., Colosimo, A., Greenwood, C. a n d Wilson, M. T. (1977) Pzoc. Natl. Acad. Sci. USA 74. 3 1 2 8 - - 3 1 3 2 . [7] Y o n e t a n i , T_ ( 1 9 6 0 ) i_ BioL Chem. 2 3 5 , 8 4 5 - - 8 5 2 .
34
tanu~y
1978
[8] Davies, G. E. ~nd Stark, G. R. ( 1 9 7 0 ) Pzoc. l~atl. Aead. Sci. U:~A 66, 651--6S6. [9] W~son, M. T., G r e e n w o o d , C., Brunod, M. and A n t o n h t i ,
E. (1975) Biochem. L 147, 145-153. [10] G i b s o n , Q. H., G r e e n w o o d , C., W h a r t o n , D. C. a n d P , ~ n e r , G. ( 1 9 6 5 ) J . Biol. C h e m . 2 4 0 , g 8 8 894. [ 11 ] Gibsor:, Q. H. a n d G r e e n w o o d , C. ( 1 9 6 3 ) B i o c h e m . 3. 86, 5 4 [ 5 5 4 . [12] Weber, K. a n d O s b o r n e , M. ( 1 9 6 9 ) J. Biol. C h e m . 244, 4406-44t2. [13] Wharton, D. C. a n d Grfffiths, D. E. ( 1 9 6 2 ) Arch. Bfochem. Bgophys. 9 6 , 1 0 3 ~_14.
