The p uri f i e d f u m ar ate r e du c ta s e of n o il, h a s been shown to be s tru c t ur a l l y si m i l ar to s u c c i n a te d e h y dro g e n a se s from a number o f s ourc e s ( D ic k ie & W e in er,1979, W einer & Di c k i e , 1979) and r ec e n t sequence d e term i n a ti o n s on th e c o l i s u c c i n a t e d eh y dro g en ase, i n d i c a t e e x te n s i v e sequence hom o lo g ies betw een th e s e two f u n c t i o n a l l y d i s t i n c t enzymes (G uest 1 984), p art i c u l ar l y i n th e c y s t e i n e r e si d u e s o f th e ir o n - su l p h ur su b u n it . A ll s u c c i n a t e deh y dro g en ases and fu m arate r e du c t a s e s t h a t have been s t u d i e d to d a te c o n ta in a c o - v a l e n t l y l in k e d f l a v i n (8 o - [ N ( 3 ) - h i s t i d y l ] F A D ) , a c i d - l a b i l e s u lp h ur and non-haem ir o n (5 -8 mol. / mol. enzyme, H e d er ste d t & R utberg 1981, B ei n ert & A lbrac h t 1982). The non-haem ir o n and ac i d - l a b i l e s u lp h ur a r e c o n s t i t u t e d as one [4 F e -4 S ]^ ^ or [3F e-xS ] c e n tr e (t h e term HiPIP, high p o t e n t i a l ir o n - su l p h ur p r ot e i n , i s used as a g e n eri c f or t h i s c e n tr e ) and ei t h er one or two f er r e d o xi n c e n tr e s u s u a lly c o n s id er e d a s [2F e-2 S ] c e n tr e s , but one of t h e s e may be a [4Fe-4S] c e n tr e ( B ei n ert & A lbrac h t 1982, D ic k ie & W einer 1979, O hnishi jah a i.. 1976 a & b, R, Cammack, T ,O h n ish i p ers o n a l c o m u n ic a tio n s ) .
I n t h i s c h a p ter th e ir o n - su lp h ur c l u s t er s from th e R,
ooTl fu m arate r e du c t a s e ar e r e s o lv e d and th e n d e s cri b e d j j i s i t u , u sin g e l e c tr o n p aram ag n etic re s o n a nc e . The q u e s t i o n o f w h eth er t h er e a r e one or two o f th e b i n u c l e ar f e r r e d o xi n c e n tr e s p r e s e nt i s a d dr e s s e d , t o g e t h er with th e p o s s i b i l i t y t h a t one of th e s e c e n tr e s may be t e tr a nu c l e ar i n n a tur e . T his
c h a p ter p r e s e nt s e v id e n c e f or i n t er ac t i o n s betw een th e i n tri n s i c c e n tr e s , and di s c u s s e s th e r e le v a nc e of th e f in d i n g s to t h e u n d ersta n d in g o f o t h er su c c i n a t e - f u m ar at e o x i d o -r e du c t a s e s .
4 .2 Res.ult fl. .and_dijSc.us&i.Qji
4 .2 ,1 C om parison ofm em branes_.from_IIMG2. .and. JRG1Q3JL
The e l e c tr o n tr a n s p o rt c h a in dev elo p ed when £ . coLL i s grown on th e g l y c er o l / fu m ar at e medium i s r e l at i v e l y s im p le , c o n s i s t i n g o f s p e c i f i c d e h y dro g e n a se s, m enaquinone, and t erm i n a t i n g i n fu m arate r e du c t a s e , A cytochrom e o x id a s e i s a l s o pr e s e nt (In g led ew & P o o le, 1 984). The jQûü. s tr ai n JRG1031 e n a b le d th e fu m arate r e du c t a s e to be s t u d i e d i n s i t u throu g h th e a m p lif ie d e x pr e s sio n o f fu m arate r e du c t a s e . I t i s t h e i n t e n t i o n , i n t h i s c h a p ter , to r e p o rt s t u d i e s on fu m arate r e du c t a s e JLû s i t u u sin g membranes from t h i s £ . c o l i s tr ai n . An i n s i t u stu d y has a number o f a d v a nta g e s, t h e pri n c i p a l one o f which i s i n overcom ing th e v ari a b i l i t y and h e ter o g e n ei ty which h as a f f l i c t e d th e s t u d i e s u s in g p uri f i e d pr e p a r at i o n s ( f or revie w s e e B e i n ert & A lbr ac h t, 1982). The use of membranes h a s th e d is a d v a n ta g e o f th e pre s e nc e o f o t h er ir o n - su l p h ur c e n tr e s which do not belo n g t o t h e fu m arate
r e du c t a s e . Thus, i t i s n e c e s s ar y to e s t a b l i s h which e . p .r . d et e c t a b l e c e n tr e s a r e at tri b u t a b l e to fu m arate r e du c t a s e , i n or d e r t o a s c ert a i n to what e x t e n t c e n tr e s b e lo n gin g t o o t h er enzymes may i n t er f e r e with th e a n a l y s i s . The ir o n - su l p h ur c e n tr e s dev elo p ed by an £ . c o l i K12 pr oto tr o p h (EMG2), grown a n a e r o bi c a l l y on g l y c er o l / fu m ar at e medium, have been d e s crib e d (In g le d ew 1983) and so t h i s e ar li er work can s erv e as a c o n tr o l i n th e pr e s e nt s tu d y . I n t h i s e ar li er stu d y s e v er a l ir o n - su l p h ur c e n tr e s w ere d e scri b e d b u t t hr e e p red om in ated . These t hr e e were t e n t a t i v e l y a s s ig n e d to a s u c c i n a te - f u m ar at e o x i d o -r e du c t a s e on t h e b a s i s o f th e predom inance o f fu m arate r e du c t a s e i n th e membranes, and a l s o th e s i m i l ari t y , i n s e v er a l r e s p ec t s , betw een th e o b serv e d p r o p e rt i e s of th e ir o n - su l p h ur c e n tr e s and th o s e r e p o rt e d f or su c c i n a te - f u m ar at e o x i d o -r e du c t a s e s from o th er s ourc e s .
Shown i n T ab le 4.1 i s a com p ariso n o f th e m ajor p a rt i a l r e ac t i o n s o f th e r e s pir at or y c h a in s o f t h e two s tr ai n s u sed . T ab le 4 .2 shows a q u a n t i t a t i o n of th e d e te c te d ir o n - su l p h ur c e n tr e s from s tr ai n s JRG1031 & EMG2. I t i s a p p ar e nt, from T ab le 4 . 1 , t h a t th e only l ar g e change i n a c t i v i t y m easured was o f th e fu m arate r e du c t a s e . T his c orre s p o n d s with th e a m p l i f i c a t i o n o f two e . p .r . si g n a l s (H iPIP a and f er r e d o xi n I l a & b o f Ingledew 1 9 8 3 ). Thus one c e n tr e , p a ram ag n e tic i n t h e o x id is e d s t a t e , i s a m p l if i e d a lo n g w ith a f er r e d o xi n - l i k e c e n tr e r e du c i b l e by s u c c i n a t e (g^ = 2 .0 3 , g^y = 1 .9 3 ) and a f er r e d o xi n - l i k e c e n tr e redu ced by d i t h i o n i t e . A com parison o f e . p .r . s p ec tr a o f c o n tr o l and JRG1031 membranes, i s b e s t
T a b le 4.1" P art i a l r e ac t i o n s o f r e s D lr a b o r y - .
chai ns I n ETPa
£H32,
.JJG.1D31
F um arate r e du c t a s e 1.27 9 «98 NADH d eh y dro g en ase 0 .6 2 0 .6 8 L ac ta t e d e h y dro g en ase 0 ,0 8 0 .4 2 Form ate d eh y dro g en ase 0 ,0 2 0 .0 2 L-glyC “3P d e h y d ro g e n a se 0 ,0 2 0 ,0 4 Su c c in a te d e h y dro g en ase 0 .3 0 0 ,4 8 A c t i v i t i e s ar e e x p re s s e d a s /<mol, / min, / mg pr ot e i nC e l ls were grown on g l yc er o l / fu m arate medium and ETPs were made as d e s cri b e d i n M a t eri a l s and M ethods,
*
gi v e n by a com parison o f th e work h e r ei n w ith th e p u b lis h e d work on EMG2 (In g led ew , 1 983), w here s p ec tr a tak e n over a wide ra n g e o f c o n d i t i o n s can be compared. A d d itio n a l c e n tr e s t h a t ar e o b se rv e d i n th e membranes from EMG2 c e l l s ar e obscured i n JRGI0 3 I membranes, by o v e r la p due to th e h ig h er l e v e l s of th e t hr e e c e n tr e s a s sig n e d to fu m arate r e du c t a s e , and t h e c o n se q u e n t use o f low er g ain . Thus t h e s e t hr e e r e s o l v a b l e s p ec i e s can be a t tri b u t e d to f u m ar ate r e du c t a s e , and th e