(1). H ÷-transhydrogenase was measured during trains of short f lashes fired at high frequency. The yield of the transhydrogenase reaction per f lash was ha l f maximal when the t ime between the f lashes was approx. 50 ms. The max imal yield was decreased when either electron transport or the transhydrogenase reaction was partially inhibited (by e i ther myxothiazol or NAD +, res0ectively) but the f lash frequency at which half -maximal yield was reached was not affected. The ma x i ma l yield per f lash was substantial ly less than the theoretical max imum. The results are discussed with reference to the membrane proton flux and to the turnover t ime of the transhydrogenase enzyme. (2) In the presence but not in the absence of val inomycin the transhydrogenase reaction persisted for several seconds after cessat ion o f e i ther a f lash train or a short period of cont inuous i l luminat ion. Inhibit ion by nigericin and correlation of post - i l luminat ion transhydrogenase with changes in the suspens ion pH measured with cresol indicated that the reaction was driven main ly by the slowly-decaying transmembrane pH gradient.
Introduction
Nicot inamide nucleot ide H÷- t ranshydrogenase in mitochondria l and bacter ial m e m b r a n e s catalyses the t ransfer of H - equivalents between NAD(H) and N A D P ( H ) and is coupled to the protonmotive force (zip)
NADH + NADP ÷ + H ~, ~- NAD ÷ + NADPH + H ~.
where H~ and H~ signify the involvement of protons at high and low electrochemical potent ial , respectively. For reviews, see Refs. 1, 2. Chromatophores ( inverted m e m b r a n e vesicles) from photosynthet ic bacteria have an active H +-transhydrogenase [3,4]. The rate of reac- t ion from left to right can be accelerated 20 fold by a Ap genera ted by photosynthet ic electron transport . T h i o - N A D P ÷ is a good substrate for H+-t ranshydro - genase and the format ion of t h i o - N A D P H is accompa- nied by absorbance changes at 395 nm, close to an isosbestic point in the l ight-dark difference spectrum of
Rb. capsulatus [3]. Thus, the kinetics of the reaction can be moni tored in real time with resolution on the scale of 10 -3 s [5]. In this report we describe the propert ies of t ranshydrogenase in flashing light. The dependence of the yield per flash of the transhydro- genase reaction upon the dark time between the flashes provides information on the rate-l imit ing components in the overall reaction: if the dark t ime is too short the rate-l imit ing reactions will not be completed. The re- sults are discussed in the context of the rate of input of energy to the reaction , the rate of leakage of Ap and the rate of turnover of the t ranshydrogenase enzyme. By analogy with 'pos t - i l luminat ion A T P synthesis" [6] the p h e n o m e n o n of 'pos t - i l luminat ion transhydro- genase ' is also described in this report. As in [6], post- i l luminat ion t ranshydrogenase was p r o n o u r ed only unde r condi t ions in which membrane poter ,I, normally the major contr ibutor to Ap in chromato- phores, was replaced by ApH. The reason for this is discussed.
Methods
Correspondence: J.B. Jackson, School of Biochemistry, The Univer- Rhodobacter capsulatus strains N22 and 37b4 were sity of Birmingham. Birmingham, BI5 2TT, U.K. grown and chromatophores were prepared and assayed
for bactcr iochlorophyll content as dcscr ibcd [7]. Ab- sorbancc changes wcrc rccordt:d in a laboratory-con- s tructed single-beam spectrop! totomctcr . Short per iods of cont inuous i lhtmination were provided, under con- trol of the computer , with a high-powcr l ight-emit t ing diode, maximum emission, 880 nm, half band-width, IIX) nm [5]. Saturat ing flashes, half peak durat ion ap- prox. 10 ~us, from a xenon discharge tube, were trig- gered by pulses from an 8DO module of a Microlink (Biodata, U.K.), also under software control.
R e s u l t s a n d D i s c u s s i o n
Transhydrogenase qfier short flash ercitation Even after extensive signal averaging, wc have bcc.~
unable to measure H ' - t r a n s h y d r o g c n a s e activity af ter a single light flash that is short enough and intense enough to drive single charge separa t ions through all react ion centres in a chromatophore suspension: the react ion yield is evidently too low (sec below). How- ever. it was possible to measure the t ranshydrogenase react ion during trains of s ingle- turnover flashes. Fig. IA shows a recording of the N A D H - d e p e n d e n t reduc- tion of t h io -NADP ÷ by chromatophores before, during and after a train of flashes fired at high frequency. Al though the amount of t ranshydrogenase react ion after each flash was not separa te ly resolved under
Flosh,og stops
Flashing starts
Fig. i . Transhydrogcnase activity dur ing trains o i short flashes. Chromatophore ,~ f rom Rh. ~,'ap,vld~lllts s t ra in 37b4 were s u s p e n d e d in a m e d i u m con ta in ing 10~ sucrose , 51) m M K ' - T r i c i n e (pH 7.6), 30 m M KCI. 2 mM MgCI 2. 0 . 2 t a g / m l ventur ic id in , 1.0 # g / m l r o t e n o n e to a bac te r ioch lorophyl l c o n c e n t r a t i o n of 10 ~ M in a vo lume of 3.1) ml a n d incuba ted at 30 ° C for 5 rain in the dark . Nuc leo t ides were a d d e d (130 /,tM N A D H and 6h taM l h i o - N A D P + I a n d the flash t ra in was t r iggered at a f r equency of 33.3 t tz . The t race shows a b s o r b a n c e c h a n g e s at 395 nm due to the fo rma t ion o f t h i o - N A D P H co r r ec t ed by sub t rac t ion for b a c k g r o u n d abso rbance c h a n g e s d u e to c h r o m a t o p h o r e p igmen t s r eco rded in suspend;ions in the absence of nucleot idcs . T h e exper iment in t r ace A was pcrfl~rmed wi thout f u r t he r modif ica t ion , in t r ace B val inomycin (ILl ta M) was p resen t .
/ s ~_ •
/ -
l@ i i i [ ; ' ~ _ z i ~ _ _ 0 100 200 300 t.O0 SO0 600 700 800 900 ~000
DQrk Time Ires)
Fig. 2. Tile d e p e n d e n c e o f l r a n s h y d r o g e n a s c yield per f lash on the da rk t ime be tween f lashes in a t ra in. Exper imcn*s were p e r f o r m e d (in the a b s e n c e of va l inomycin) as de sc r ibed in Fig. i. T h e yield was ca l cu la t ed f rom the slope o f the l inear r a t e a f t e r approx . 5 f lashes a f t e r the onse t o f the t ra in . The e r r o r ba r s show the limits o f accu racy of the yield based on a subject ive impress ion o f the maxi- m u m and m i n i m u m . s l o p e s o f the da t a tha t we re likely given the s i g n a l / n o i s e rat io, e . No f u r t h e r addi t ions , A plus 0.1 ~ M ca rbony l
cyanide p- t r i f l u o r o m e t h o x y p h e n y l h y d r a z t m e .
these condit ions, the yield pe r flash could be ca lcula ted from the slope (cor rec ted for the ra te of react ion in the dark) divided by the flash frequency. The yield of the t ranshydrogenase react ion per flash was independen t of the number flashes, as shown by the fact that the appa ren t rate o f t ranshydrogenase dur ing the train was almost constant .
The d e p e n d e n c e of the yield of the t ranshydro- genase react ion per flash on the dark t ime be tween the flashes in a train is shown in Fig. 2. The maximum yield per flash was observed when the dark t ime was grea te r than about 150 ms. The calculat ion of flash yield became increasingly inaccurate at dark t imes grea te r than about 1000 ms, as the slope of the da ta during the flash train became barely dis t inguishable from that in the dark period, if pro ton leak across the ch romatophore membrane were negligible, then the inward pro ton cur ren t gene ra t ed by photosynthet ic e lec t ron t ranspor t should be precisely matched by the outward pro ton current through t ranshydrogenase . Thus, assuming that H + / H - = 1 for t ranshydrogenase [8,9], that H + / c - = 2 for photosynthet ic e lec t ron trans- por t [10] and that there is one photosynthe t ic react ion cent re per 100 bacter iochlorophyl l [9], then at increas- ing dark t imes be tween flashes to permi t each e lec t ron t ranspor t react ion to turn over complete ly , the yield per flash should approach a maximum of 0.02 thio- N A D P H per bacter iochlorophyl l . Tha t the observed maximal yield (Fig. 2) was only about 10% of this value suggests that only a small fraction of the pro ton cur- rent genera ted by photosynthet ic e lec t ron t ranspor t was used to drive the t ranshydrogenase reaction. This conclusion is quant i ta t ively suppor t ed by direct mea- surements of membrane ionic current in the presence
and absence o f t r anshydrogenase subs t ra tcs [9]. In the context o f the s imple scheme,
elec t ron t ranspor t , A p f
t r anshydrogenase
" ' l eak"
Scheme I
we conc lude that the ra te of e l ec t ron t ranspor t (and assoc ia ted p ro ton up take ) and the ra te of p ro ton leak- age are re la t ively high and that the ra te of H ÷ efflux accompany ing the t r anshydrogenase reac t ion is re la- tively low. Note tha t the compe t ing leakage current , though it is compara t ive ly large, p robab ly does not s ignif icant ly inhibit H +- t ranshydrogenase in high inten- sity con t inuous l ight because the m e m b r a n e po ten t ia l r eached u n d e r such condi t ions is sufficient a lmost to sa tu ra te the reac t ion [9]. The ident i ty of the compo- nent(s) carrying the ionic cu r ren t in the absence of nue leo t ide subs t ra tes has not been es tabl i shed . The expe r imen t s desc r ibed in this r epor t were rout ine ly ca r r i ed out in the p resence of ventur ic id in to inhibit F0 o f the A T P synthase. T h e to ta l m e m b r a n e ionic cur- ren t was only sl ightly a f fec ted ( < 30%) when C I - was r ep l aced by SO42- o r Tr ic ine o r when K ÷ was rep laced by Na + o r t e t r a m e t h y l a m m o n i u m (da ta not shown) .The effect o f the passive m e m b r a n e conduc tance on the yield of the t r anshydrogenase reac t ion is less pro- nounced at high flash f requenc ies where the genera t ive cu r ren t is large, as shown by the effect of uncoupl ing agent (Fig. 2). Thus, at high flash f requencies , 0.1 /.tM carbonyl cyanide p - t r i f l uo romethoxypheny lhydrazone had only a small effect (32% inhibi t ion) on the flash yield bu t it was more inhib i tor , ( > 80%) when the da rk t ime be tween f lashes was gre Jter than 200 ms.
If the diss ipat ive ionic cur ren t s were less p ro- nounced , then da t a such as t h o : e in Fig. 2 would yield the tu rnover t ime for e lec t ron t ranspor t -d r iven trans- hydrogenase and specific inhibi t ion o f whichever pro- cess were ra te - l imi t ing (e lec t ron t r anspor t or t rans- hydrogenase ) would more s t rongly affect the f requency d e p e n d e n c e . In fact, ne i the r inhibi t ion of e lec t ron t r anspor t with myxothiazol no r inhibi t ion of t rans- bydrogenase by N A D + (a reac t ion produc t ) signifi- cant ly a f fec ted the f requency d e p e n d e n c e , a l though bo th dec rea sed the maximal yield p e r flash (Figs. 3 and 4). I t seems likely, in view of the commen t s above, that the reason for this is that the f requency d e p e n d e n c e is d o m i n a t e d not only by tu rnover of e lec t ron t r anspor t and t r anshydrogenase , but also by the passive conduc- tance of the e h r o m a t o p h o r e m e m b r a n e . Thus, over a
_ . 5
o ,'7"
1 SO & o o
= t 25 o
.~ 1 O0
::x 0 75
0.50 l o z :
-- 0 25 o
p u s 2s~M ~ * l o ~ , , ~ z c l
• a .
.
plus SO~M ~¥x0tt~,atol
I l 100 200 300 ~.00
-1
DQrk hme between floshes (msl
Fig. 3. The effect of nl.vxothiazol on the relat ionship be tween trans- hydrogenase yield p e r flash on the dark t ime be tween flashes in a train. Exper iments were p e r i o r m c d a-: descr ibed in Figs. t and 2. o , No fur ther addit ions: o. plus 25 nM myxothiazol; • , plus 51) nM
myxolhiazol.
range of flash f requencies inhibi t ion of e i ther the elec- t ron t r anspor t c ompone n t s or of the t r anshydrogenase enzyme can lead to depress ion in the rate of the overal l react ion. The a p p a r e n t half- t ime of 50 ms for tu rnover o f e lec t ron t ranspor t -d r iven t ranshydrogenase (Figs. 2 - 4 ) does, in fact, closely co r respond to the value of k~,, for t ranshydrogenase [5].
Venturo l i and Mclandr i [11] showed in ana logous exper iments , also in ch roma tophore s , that the degree of inhibi t ion of A T P synthesis by e i ther dicyclohexyl- ca rbod i imide or by ant imycin A was almost indepen- den t of flash f requency - there was c lear cor respon- dence with the results descr ibed here for H+-trans-
•o• 1-50
1 25
.~ 1 O0 o E
z , 0 50
o .1=
0.zs
o o c~ NO ~ l h 0 n s
0 0 •
o o o • • • p lus 6 ~ u M N A D °
0 • • • -&
• • • plUS ~.mM NAD"
I I ...... I
100 200 ~00 ~00
Dark time be,ween floshPs ms}
Fig. 4. Th e effect of N A D ~ on the d e p e n d e n c e of t ranshydrogenase yield per flash on the dark t ime be tween flashes in a train. Experi- m e n t s were p e r fo rmed as descr ibed in Figs. I and 2. c~+ no fur ther
additions; o. plus 6110/.tM N A D + ; A, plus 4 m M N A D +.
24
hydrogenase. -l-hose u, orkcrs, ho,.,.evcr, concluded that ATP synthesis correlated svith the nuntber of active electron transport chains or :iclivc ATi" s.vnthases inde- pendent ly of the flash frequency, qhcy suggested that the results indicate that lhc energetic mtcrmcdia lc in photophosphoryhtl ion does not dclocalisc and there- forc cast doubt on the involvmcnt of the (dclocaliscd) protomotivc ti}rcc. This conclusion ~as based on the argument that a change in the flash frequency shifts the position of the rate-limiting step in ATP synthesis and therefore alters susceptibility to specific inhibitors. In fact, it is likely that the flash train profiles of A TP synthesis in chromatophores are also profoundly influ- cnced by dissipative leaks: the maximum yicld [11] wits t).(~)ll ATP per bacteriochlorophyll (which, assuming l t ' / A T P 3 [7], | | * / c = 2 and I photosynthetic reac- lion centre per I00 I~acteriochlorophy!ls) represents only lbr~ " of the theoretical value (expected in the absence of leak), il the leak is significant, and accept- ing that the rate of the kmic leak is non-l inearly dependent on the value of the protonmolivc force [7], then the dependence of the extent of inhibit ion by antimycin A and by dicyclohexylcarbodiimidc on flash frequency is extremely difficult to predict.
In the presence of va[inomycin, the kinetics of the t ranshydrogcnasc rc,'tction dur ing the flash train changed substantially (Fig. 1B). At the onset of flashing there was a lag in the rate of reaction before steady- state was approached, some 3 s later. The durat ion of the lag increased at decreasing flash frequencies (not shown). The presence of a lag can probably be ex- plained (see below) by the slow development of ApH under these condit ions in which A ~ is collapsed by valinomycin-catalyscd K'-efflux. What is probably an analogous lag at the onset of ATP synthesis by chloro- plasts during flash excitation has been examined in depth with somewhat conflicting results [12-16]. Be- cause the rate of the t ranshydrogenase reaction was not constant in the prcsencc of valinomycin, the depen- dence of flash yield on frequency wits not investigated. Fig. IB also shows that at the end of the flash train the transhydrogenase reaction cont inued for a substantial ly longer period than in the absence of valinomycin. This phenomenon will be called post- i l luminat ion trans- hydrogenase, it was studied in more detail using short periods of cont inuous i l lumination.
Post-illumination transhydrogenasc it was shown [9] that, in unt rea ted chromatophores
of Rb. capsulatus, periods of photosynthetic i l lumina- tion led to an increase in the rate of H+-transhydro- genase activity and that upon extinguishing the light the ra~e reverted to the original dark level within a few hundred milliseconds. Fig. 5A illustrates these points except that on this rather slow time-scale the kinetics of the return to the dark level after i l luminat ion were
OFF
~ No ndd~hcns
/
/ /
B~-J .........
/ '~ Dlus ~,tlhnomy(,n 7 St/o J C J and n,g ...... ,.~T .... l
? [I~HT
~N
OF~
q I
O_.j ~No~__._____.ad~,ttoos
~?omyc*n
I LtCJMT
ON
plus vohnomyon Qnd nige?ffln
1 150
Fig. 5. l " t a n s h , , d r o g e n a s c r a t e s a n d d e v e l o p m e n t of m e m b r a n e po - ten t ia l d u r i n g and a f t e r sho r t pe r i ods o f c o n t i n u o u s i l lumina t ion . C o n d i t i o n s a~ Fig. I except t ha t the ¢ h r o m a t o p h o r c s were p r e p a r e d f rom Rh. cap.w#h+tus s t ra in N22. K ' - T r i c i n ¢ was r e p l a c e d by Na +- Tr ic ine and the r eac t ion vo lume was 2.5 ml B e t w e e n the a r r o w s the chromatophorc,, were illuminated with the light.cmining diode. IA- (') Measurement of the transhydrogenase reacthm at 395 nm (traces wcre c o r r e c t e d fiw absorbancc ebb, ages occurring in the absence of nucleotide): tD-F) electrochmmic absorbance changes at 503 nm+ (A) and (D), no further additions: (B} and (E), plus 0.1 ptM valino-
m vein; ((') and (F). pltr~ x alinomycin plus 0.3 p,M nigericin.
barely resolved Upon t rea tment with val inomycin the dark rate was unaffected, the rate dur ing i l luminat ion was depressed by approx. 3 2 ~ (relative to the control) but, remarkably, the H-Ltranshydrogenase react ion cont inued at an elevated rate for many seconds after the light was switched off (Fig. 513). The post- i l lumina- tion t ranshydrogenase was maximal at about O.l tzM valinomycin (Fig. 6) and 40 mM KCi (Fig. 7). Inhibi t ion by higher concent ra t ions of KCI is a reflection of the deleterious effect of cations on the activity, e i ther in the forward (Fig. 7) or reverse direction of the enzyme in m e m b r a n e - b o u n d form [9] and in the solubilised state [17].
in the condi t ions of Fig. 5B, eleetrochromic ab- sorbance changes indicated that the average value of the membrane potent ia l ( d ~ ) dur ing i l luminat ion was depressed by about 70% relative to the control (Fig. 5E and D). When it was the only cont r ibutor to Ap, depression of At/-" by 70% caused a 70% inhibi t ion of the t ranshydrogenase rate [9]. Since the t ranshydro- genase rate dur ing i l luminat ion was actually inhibi ted by only 32% by valinomycin, it is concluded that the decline in Ag r was substantial ly cotnpensated by the enhanced ApH expected unde r these condit ions. Thus,
2
o
/ r
0 0001 i
0 f01 0 I I ~0
C0n(enfrahon of vohnomyon ( pHI
Fig. I~. T h e d e p e n d e n c e o f post- i l luminat i (}n I ranshydrogem~se on the c o n c e n t r a t i o n o f valinom3"cin. E x p e r i m e n l s 'a, e re per f l~rmed as in Fig. 5 excep t tha t t he va l i nomyc in c o n c e n t r a t i o n was var ied as shown a n d the i l l umina t i on t ime w a s 2.0 s. Pos l - i l lumiml t ion l r a n s h y d r o g e n a s e was m e a s u r e d as the e x t e n t o f the r e a c t i o n d u r i n g the t0 s p e r i o d a f t e r swi tch ing o f f the l ight , c o r r e c t e d for the r a t e of the l r a n s h y d r o -
g e n a s c r e a c t i o n in the init ial da rk pe r iod .
it is possible that an e levated value o f J p H in the presence of val inomycin is also mainly responsible for the increase in post- i l luminat ion t ranshydrogenase ac- tivity. In support o f this, Fig. 5C shows that post-il- luminat ion t ranshydrogenase was strongly inhibited by 0.3 # M nigericin, which depresses J p H through its K * / H ~ exchange activity. Nigericin had a less pro- nounced inhibitory effect on the t ranshydrogenase rate dur ing i l lumination, probably because it caused a slight compensa tory increase in the value of ,1'/" (Fig. 5F). Note that in the presence of nigericin, J V col lapsed more rapidly after ext inguishing the light (the result of dissipation o f the diffusion potent ia l gene ra t ed by K * efflux in the presence of valinomycin a lone) and this too would cont r ibu te to inhibit ion of post- i l luminat ion t ranshydrogenase .
The d e p e n d e n c e of post - i l luminat ion t ranshydro- genase (in the presence of valinomycin and K 4 ) on the length of the light period is shown in Fig. 8. The largest
o
o °
E __=_
Con(enfr~xtion KCI (mH)
~ L
o o
~ g
Fig. 7. T h e d e p e n d e n c e o f pos t - i l l umina t ion t r a n s h y d r o g e n a s e on the KCI c o n c e n t r a t i o n , E x p e r i m e n t s as in Fig. 5B excep t t ha t the KCI c o n c e n t r a t i o n w a s va r i ed as shown a n d the i l lumina t ion t ime was 2.0 s. Pos t - i l l umina t ion t ransh~. ,drogenase w a s m e a s u r e d in the 10 s pe r iod a f t e r swi tch ing o f f the l ight, c o r r e c t e d for the r a t e of t r ans -
hydn~genase in the da rk ,
25
2
c~ o
~ ° E
.L
2 0
t i
Time of lllLJmln~hen (5)
F i g . 8. T h e d e p e n d e n c e o f p o s t - i l l u m i n a t i o n t r a n s h y d r o g c n a s ~ - on t h e
time of illumination and correlation with light-dn,,cn prohm uptake. o, experiments as in Fig. 5B except that the time of illumination was ~,aricd as shown. Po~t-illumination transh~drogeuasc v, as measured in the I() s period after switching off the light, corrected l~}r the rate of Iranshydrogenasc ill the dark. ,. experiments as in Fig. 513 except thai nt) Tricine was present. The medium was supplcmentcd with 100 ~ M c~csol red and adjusted to pH 7.6 by comparison v.ith the ahsorhauce of u buffered control. Absorhance changes al 572 nm during illuminalion reflect the p l l of 1he chromalophorc ~uspension. ]'he data points give the extent of II " uptake as a function of the
time of illunfination.
increase was observed af ter 1-2 s of photosynthet ic irradiation but the extent of the process was still in- creasing slightly even after 5 s. Also shown in Fig. 8 is a correlat ion be tween these data and the kinetics o f l ight- induced proton uptake (in the presence of valino- mycin and K ~ ). It was demons t ra ted separately (not shown) that valinomycin enhanced the extent of light- induced proton uptake by approx. 5-fold, the result of a decrease in AV due to e lec t rophore t ic efflux of K*. Since the buffer ing capacity of the chromatophore lumen is only weakly dependen t on bulk phase pH in the range pH 5 - 7 [18], the correlat ion between the dependence of post- i l lumination t ranshydrogenase and of H*-up take upon the t ime of i l lumination fur ther supports the view that J p H is the major driving force in the process.
We conclude that not only AV [9] but also J p H can drive t ranshydrogenase. The larger storage capacity of the la t ter relative to the former explains why significant quant i t ies o f post- i l lumination t ranshydrogenase arc observed in the presence and not in the absence of valinomycin. Al though / IV and J p H are thermody- namically equivalent components of the protonmot ive force, differences be tween their ability, away from equil ibrium, to drive the t ranshydrogenase react ion may be instructive in unders tanding ~he react ion mech- anism. If we assume that an increase in A p H compen- sates completely for the decl ine in / tV during the l ight-driven t ranshydrogenase react ion in the experi- ment shown in Fig. 5B, then using the known relat ion- ship [9] be tween t ranshydrogenase rate and J V (for J p H = 0 ) , it can be calculated that J p H is approx. 50% as effective as J V in driving the reaction. Since
2t~
ApH compensat ion is expected .o bc rather less than complete, thi~ is an u n d c r c s t i m d c and il is not incon- ceivable th; t the two componet, ts are kinetically cqttJv- alcnt in driv!ng the translaydrolzenasc reaction.
Aeknowledgem,~nt
This work wa:; :;upporlcd hy a grant from the Sci- on:c" and Engineer ing Rcscawch Council .
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