The function of plastoquinone as electron and proton carrier in photosynthesis

Bioelecfrocizemistry

and Bioenergetics

3, 302-318

(w76)

The Fu@ion of Plastoquinone as Electron and Proton Carrier in Photosynthesis * by U. SIGGEL Max-Volmer-Institut.

Technische

Zt’niversiti&

Berlin,

FRG

Abbreviatiohs proton translocator molecule of unknown identity reaction centre chlorophylls of photosystem and Chl-an, respectively cytochrome f W-f DCMU 3-(3.4-dichlorphenyl)-1.1-dimethylurea n-(N-morpholino)-ethanesulfonate MES PCY plastocyanine plastoquinone PQ plastohydroquinone PQH, plastosemiquinone PQHPQ+pfastosemiquinone-anion sum of these 4 species PQo Tricine N-tris (hydrosymethyl) methylglycine U direct acceptor for the electrons of the PQ-pool (mainly not included in the pool special plastoquinone, X special plastosemiquinone-anion Xtwin of two XX2’sum of twins of Xand X X*0 primary electron donor of light reaction II T primary electron acceptor of light reaction I Z

AH Chl-aI

I

and

II,

PCy)

The aim of this paper is to compile earlier and recent experimental results by flash photometry in order to discuss the possible mechanisms of coupled proton and electron transport by the plastoqninone system. The results from different techniques (e-g_ fluorescence studies) which _ ___ sometimes lead to different conclusions are not included. * Presented at the 3rd International Jiilich, 27-31 October 1975_

Symposium

on Bioelectrochemistry,

Plastoquinone

as Electron

and

Proton

Carrier in Photosynthesis

303

The redox reactions of plastoquinone can directly. be shown by flashinduced absorption changes which are dependent on the electron transport inhibitor DCMU. A new interpretation for the reaction constant kd, involved in the reduction of PQ, is given (different from that in Ref. 3)_ The kinetic equation for the o_xidation of plastohydroquinone is confirmed_ For the constant of oxidation k, in addition to the previous interpretation (Ref. 3) a different one is proposed corresponding to the concept of special reaction sites as opposed to that of homogeneous reaction kinetics_ 3 The proof for the involvement of P& in the proton transloeation is based on the assumption that a proton pump will give rise to a pHdependence. Apart from the measurements on chlorophyll-ni as direct evidence the pH-dependent oxidation of PQH, is presented. 3- For the esplanation of the pH-dependence the concept of kinetic control realized by some unknown relais mechanism is put forward, The identity of the molecule deliverin g the protons into the inner phase of the thylakoids (plastosemiquinone or a special translocator molecule) is dicussed. 4 Taking the interpretations of the pH- and DCMU-dependent effects together, two extreme models for the P&-pool, the fluid (fluid phase) and the statistical model (possibly mesomorphic phase) are treated quantitatively ; the latter is favoured. I.

d.

Introduction In the photosynthetic electron transport of higher plants, electrons are transferred from water to a final electron acceptor_ This process is driven by two light reactions which result in a charge separation around two special -molecules of chlorophyll, designated as chlorophyll-al and chlorophyll-air. Plastoqtinone is localized between the two photosystems (Fig; I). Its light-induced redox-reactions may be visualized directly via the difference between the absorption of the quinone Pg and th_at of the hydroquinone PQH,, being maximal at 365 nm_1s2 Following a short flash of IO-~ s PQ is rapidly reduced (assuming a half-time of 0,6 ms) by electrons delivered from light reaction II. The subsequent reoxidation by positive redox equivalents from light reaction I is much slower ; a$ pH = 7 it proceeds with a half-time of about 20 ms3 (somewhat slower in Fig. g top)_ This reaction, in which electrons are transferred from plastohydroquinone to plastocynine, PCy, and cytochrome f, Cyt-- (both designated as U in Fig. I), is the rate limiting step in the electron transport chain. A long flash of IOO ms induces a more extensive reduction of PQ, indicating that it is present in a higher concentration than the other electron carriers : 6-10 molecules per centre 11.3=4 The stationary degree of reduction is determined by the rates of electron influx and effluxAt high light intensities appro,ximately half of the pool is reduced. The

Siggel

304

PO01

antipool

1

tZe

Chl-aI-U

i f-

Chi-aI++-

U t-

Z f-

CM-aIt_

U f-

Z t-

thl-aI’t-

U t-

C-.

e-

x ~:_iJG$; Y C-H, 0 -__--*

f-

X :I-&i:+; ---____-x <;Jr
Y cy

H20 blocked by OCMU

Simplified scheme of photosynthetic electron transport showing the cooperation of eIectron transport chains by the combined pools of plastoquinone- Inhibition of photosystem II by 3(3.4-dichIorphenyl)-r.x-dimethylurea (DCXIU).

halftime of reduction is dependent on the light intensity and varies from 6to15ms(for3xro53 x 106 erg cm-’ s-l) _ The dark osidation exhibits an only partly irreversible kinetics, The reversible part with a half-time of I+ZO ms, corresponds to the electron capacity of three of the antipool, consisting of chl-csr, Cyt-f and PCy (Fig. z)_ Complete oxidation may be achieved by the addition of appropriate electron acceptors (fenicyanide) or by a far-red back,oound light (720 run) esciting only light reaction I. The plastoquinone molecules of 10-40 chains represent a functional unit_~ It is possible to block go o/0of the centres II by an inhibitor without chanting the number of reducible chlorophyll-+ molecules. Separate electron transport chains do not exist. The common PQ pool (strand) accepts electrons from a number of reaction centres II in order to deliver them to a number of centres I which is not necessarily the same.

l--+-l

(

____-----_T_r_----

:~~2

/ t

~~ I

I 0

t 50

9 100

, 0

P (ms.1

50

t 100

Fig. a. Absorption change at 265 nm induced by a Iong Aash : reduction of plastoquinone in the light, reosidation of the hydroquinone in the darkBentylviologen 1.6 XIO-~ df, NH&l 3 x10-2 X. pH = 6.5_

Plastoquinone

as Electron

and

Proton

Carrier in Photosynthesis

30.5

The accurate measurement of the PQ-absorption changes in the ultraviolet region is difficult especially at a low pH_ Therefore it is worthwhile to look for an indirect method. All electrons from the pool finally arrive sometime at the chl-nr-centres : moreover, after a short flash, this is true for the time of a kinetic trace for go o/oof the electrons.6 The oxidation of P&H, is the kinetic counterpart of the reduction of o,xidized chl-nr after a short flash.3 The oxidation of PQH, can obviously only begin after its formation, so that a time lag in the reduction of chl-aI+ is to be expected. Because of the large difference in time constants this lag is, however, small. Besides it can be observed only after an appropriate dark period. Fig_ 3 shows the time la,= as well as the characteristic 20 ms time. The qualities of PQH, may thus be deduced from chlorophyll-err measurements at 703 nm which is much simpler.

Fig_ 3. chlorophyll-a, Reduction of oxidized a time after a short flash, exhibiting lag. Top : pH = 6,s ; bottom I pH = S_

cl

40

80

12G T

160

Measurements of chl-ar can also be performed in the absence of an uncoupler of phosphorylation. In this case a light-induced alkaliniza- ’ tion of the suspending medium takes place. Protons are pumped into the inner phase and establish a pH-gradient across the thylakoid membrane.’ Two protons per electron are translocated.8 Since the introduction of the chemiosmotic theory, it has been assumed that PQ accepts protons from the outer phase after reduction, in order to release them into the inner phase during the oxidation of hydroquinone. The location of the proHowever, the tolytic sites in relation to the membrane can be shown9 direct proof of the involvement of PQ has not been possible until now. The properties of P& as a earner of electrons and protons will be considered in more detail here.

S&e1

306

Experimental

The absorption change of chlorophyll-nr was measured at 703 nm with the single flash technique, that of plastoquinone at 265 nm with the repetitive flash technique_ Spinach chloroplasts, stored in liquid nitrogen and thawed before measurement were used. Standard reaction rnisture I pro mild buffer of various pH (MES for pH < 7. Tricine for pH 2 7). 50 mM I
Plastoquinone as electron carrier Redzrctio-rt of PQ

PQ seems not to be the direct electron acceptor of light reaction II. The substance X has been found as intermediate on the basis of absorption changes. lo The rate of formation of S- is very high-l1 The reoxielation, that is the transfer of electrons to PQ, proceeds with a halftime of 0.6 ms. O;-iginall~. S- was identified3 with the plastosemiquinone-anion PQ*-, an assumption supported today by the fact that the main positive peak of the difference spectrum at 320 nm coincides with that measured for PQ-- Gt vitro.12 On the ‘other hand this assumption causes difficulties in creating a model for the PQTreactionsx3 and for the interpretation of osygen measurements_ la So It should be a special plastosemiqtinone \:?th properties differing from those of the pool-semiquinone. When the electrons have entered the pool, hydroquinone is quickly formed. The mechanism is unknown. Especially there are 1x0 hints at which step the protons are accepted. According to the dissociation constants of simple benzoqninones the protonation should be complete_ The double reduced PQ is also formed after a short flash. For the dismutation of the semiquinone, which has to be the first reaction product, a cooperation of at least two electron transport chains is necessary. In fact a cooperation of chains and moreover of two reaction centers has been found-l” Based on earlier calculations3 the following forniulation is proposed for the reduction of PQ, where ~t&o~~ represents the relative number of active centers II : d [_&“-] dt-

=

Plastoquinone The

formation

as Electron a n d ,Proton. Carrier in.Photosyfi~e~s~!/--~3~bT? r a t e of P Q 2 - a t

is g i v e n b y : [d[PQ2-JW d~

~ " k rvs-1/[PQ] -

[PQo]

the reduction

-: ~ =~i!:~

side. 0 f the:~PQ ~p06ili

>j =(;"!~(:!: ~:::-:~ ,

/~=? :.:~i:~::~i:/!i

~ : : :~":: [r~):oi-.-i~n:::~--::-)-:r.~--.a~|[PQif~J:-fP~] -

. .......

n n ° ':- ...."........ :.:=- P Q o ]

::~-~'

B y a c t i o n of l i g h t (reaction Constartt k,) ~.two.imolecul~es~:0f:)a~spe~_ p l a s t o q u i n o n e X a r e r e d u c e d to the. c0_rr_esponding!semiquinone+:ani0ns!X-%:: T h i s t w i n / X , - -~.r e a c t s w i t h t w o : m o l e c u l e s i0f; ::p!ast0qt-'tin 0 h e :in--:ith@:~pool) i n a d o u b l e electron t r a n s f e r . r e a c t i o n : ( r e a c t i o n - c 0 n s t a n t . kd) p r o d u c i n g two p l a s t o s e m i q t f i n o n e - a n i o n s , w h i c h , subsequently)-dismutate-:~etyi:~fas~ -A b a c k f l o w of electrons f r o m t h e pool. to t h e .speci~tl p l a s t 6 q u i n 0 n e at~4:he r e d u c t i o n side (reactionconsfant~k-h)~ is~formt~lafi~daccord~ng.~to!theo~:: d a t i o n , r e a c t i o n a t - t h e o x i d a t i o n s i d e 0f-.the pool._ ~--~--:.:i~;--.-:~.~. :_~;-:i.-i_:-:-~~ !:~:.W i t h t h e a p p r o x i m a t i o n o f B O D E N S T E I N ~[X~*~-] i s found:i.to..bei'~ ::- : [ X ~ - ] = D~. o] nn " k, + k,~{[PQ~-]:[PQ]/[PQa]Z}.Z--~ , ::"-_. nn o k , --F k ~ { [ P Q Z = ] [ P Q ] ] [ P Q o ] * } = + - . - k d : { [ P Q ] I [ p Q o ] } * : T h e - r a t e of " P Q - r e d u c t i o n . c a n t h e n : b e :writ~en-~do,~i-explieifly~-i F o r e x t r e m e l y h i g h v a l u e s of t h e r a t e c o n s t a n t ki (this is:probably;.!.Unreffl~. istic for a long flash] we o b t a i n t h e f o r m u l a t i o n of Ref. 3 - ( g i V e n : f d r . t h e original i n t e r p r e t a t i o n of ka) :: i.- ~:.. : " : ~. . =..":~ ii !.:_.~.::- : -. -.~:=:- ~(~:: I~ i~i.~

T h e time. c o u r s e - o f the: r e v e r s i b l e : p h a s e ) ~ a f ~ ~ r k ~ d a t i o n : ) " ~ a e ~ ) i ) ~ . l o n g fllashfits i n t o a n e x p o n e n t i a l funcfioff:;(Fig.'::Z)::-~Iti:igjddtei~mind~Tn6t. oPJ3r b y P Q b u t alSo b y . t h e i q u a l i t i e s . o f ~ t h e i ~ 6 o i n i ~ ~ i 6 f ~ i t t i e : ~ t i P ~ d L ; : T h e r e f o r e i t : i s m o r e a p p i 0 p r i a t e / t o evalua~e.-i!tlie:j~tiM!::~fd.~i~f o ~ b @ a t t h e o n s e t of. darkness:. ~. I f t h e a m o l m t 6 f ~-~tati6ff~--;t/ydroquinone:is ~. v a r i e d by_ i n c r e a s i n g i n h i b i t i o n i : p f c e n t f e s : ~ : ; ! ! ~ : ~ t . h ! - ~ D - C ~ , ! ) ~ h e - f o l l o ~ g ~ : k i n e t i c e q u a t i o n for. O x i d a t i O n - i a t e - o f . " P Q H 2 ~ a ~ - : t h 6 0 ~ X i d a t i o n s i d e of:-: t h e Q - p o o l m a y b e d e r i v e d (Fig.-4) :' :-:- :~:-: ;~::.-:::::,.-~::::-.:::=~::..-~:.~: ::--. ::~:4.:~:-:-:c d [ -Qtt d

• - .: ._.

.... -~. . . . .

:- -:..:.~-~....... -..........=....... -.:. •.......

T h i s result, C o n f i r m i u g t h a t ~of sTiE~~c[::.Wi~_~?::!.~Ko~/haa:varied.: [PQHo] b y f l a s h g r o u p s o f : d i f f e r e n t ldnds,~:hoids fox:. apH:~)6,$::~.:~.::~.:?-:..: : ; I f t h e c o n c e n t r a t i o n of "s e m i q u i n o n e ):is i l o w , ~-th-en =-the =-eXpregsi0Ii-:m e a n s t h a t t h e v e l o c i t y of t h e react]oniis-prbportional:t0::~.theiproduc[/Of-:/ t h e c o n c e n t r a t i o n s of q u i n o n e a n d h y d r o q u i n o n e Z :: T h i s : h a s to.-~be!~inte~

Siggel

Representation of the kinetic equation for the osiThe initial rate. of dation of plastohydroquinone. dark osidation xias measured as a function of the degree of pool-reduction at pH = 6.5_

preted as showing the formation of semiquinone as the first step of PQH21 oxidation_ Nevertheless, the mechanism is far from being solved_ There are at least two possibilities for the interpretation of the reaction constant k,. I_ Assuming that the PQ-pool obeys the kinetics of homogeneous solutions in its reactions, ‘then k, is the constant of commutation.3 z_ It is perhaps more realistic to regard the pool as a mesomorphic phase with translational diffusion only possible in the two dimensions of the membrane’s plane. A reaction of PQH, with the o_xidized antipool is only possible for those molecules that are in a well-defined neighbourhood of the reaction partner. For the electron transfer to occui a special reaction site has to The probability for this situation be occupied by a PQH2-PQ-couple.13 in a pool of PQ, molecules, total, and PQH, molecules, reduced, (per reaction centre II) is given by :

where .Zrepresents the number of coupled chains (at least IO)In order to obtain the rate of oxidation, ZPI,has to be multiplied by the probability that the antipool is oxidized (U+/UO = I for stationary conditions), the probability w that the PQH,-PQ-couple is in the semiquinone state, and the reaction constant k,, between U+ and PQHI-

It is not possible to exclude one of the models without varying more 1

~_ I__

& _

\

Plastoquinone

P~~q~on~

as Electron

and

Proton

Carrier in Photosynthesis

_

as a proton 6?arrier

.309 .

It was already stated that the binding of- protons from the outer phase to some species of reduced PQ has not yet been observed_ The chances .for proving the carrespouding reIease of protons into the, inner phase are better. It may be expected that, by sdme reverse action. of the proton pump, a coupled electron transfer ,reaction will be. slowed .do>vn by high proton concentrations.. Furthermore,. the oxidation of plastohydroquinone may be examined via the absorption changes of &l&o.-. phyll-nr. The pH-dependence of the reduction‘of oxidized chl-ar by electrons from the pool has been mkasured in the .pres&ce of gr&m@idin i,n high concentration to assure that internal and external pH are equaL7 As’ expected, the reaction is much slow& at a.low pH (5) than at a high pH (8) after both a long and a short Aash (Fig. 5);. The sensitivity of .the reciprocal half-time towafd pH is more pkonounced in the ~case of the After a long flash the degree of reduction of the short flash (Fig. 6). pool is higher and presumably xhanges .sqmewhat. according to the pH, leading to a pH- depefidence of r/t% which is equal to that of the stationary electron transport rate. .. : _. short

.fJ

0.4

Rash

.0,8.

I,2

Fig. 5. Time course for the r&duct& tiveiy for different external

..7,6... . .. ., of clil-~~+ after a short and

pH in the presence

of

long

gramicidia;

flash

-

r&giec~ -- ‘.

Siggel

310

with

I

0 4

6

Fig. 6. Effect of low pH I Retardation of stationary electron transport and decrease of the rate constants of chl-a,+reduction after a short or long flash (5 s)-

uncoupIer

External

1

7

pH

t 8

The external and internal pH being equal, this experiment does not prove that the pH-sensitive reaction is really at the inner side of the membrane_ This is necessary, however, for the PQ to function as a proton pump. Therefore, an experiment without uncoupler was started in which the internal phase is acidified. By increasing the -duration of- the illumination, the half-time of chl-car+- reduction in the dark is reversibly increased (Fig_ 7)_ This result would not be possible if the PQH,oxidation were situated at the outer side of the membrane where the large amount of buffer keeps the pH-value constant. The function of the PQ-system as a proton pump thus seems to be established_ For a more detailed information, a mathematical analysis of the pH-functions is necessary_ It t-urns out that the half-time of chlar+--reduction is a linear function of the proton concentration (Fig. S). Accordin& the reciprocal haIf-time is given by :

(11) Two simple interpretations are possible for this formula: I_ the expression reflects an undisturbed protonation equilibrium of a substance A, KA being the equilibrium constant; z. the protonation equilibrium of the substance A is disturbed by a successive reaction_ KA represents the ratio between two rate constants not related to the forward and back reactions of an equilibrium, and shall be called e@et%ue ~~ssoctatio~~co~astn~at. The molecule AH is the one in contact with the inner phase which delivers the proton. It may be a special proton translocator not involved in electron transport or a species of the PQ-system, possibly the semiquinone, corresponding to cases 1 and 2, respectively.

Plastoquinone

as

Electron

and

Proton

Illumination

Carrier

in

Photosynthesis

time

_.

2i? ‘:

s

5

0.1 s

ti

0

-10

20

30

40

Fig- 7_ Time course of chl-a,+-reduction without uncoupler at pH = S showing the rate decrease by internal acidification Consecutive illucaused by sufficiently long illumination. minations at the same sample.

IR

=0.2s

XJL = 2.05

1

.

s

10

Fig. S. Functional analysis of the dependence of the halftime of chl-uf-reduction on the proton concentration (with gramicidin) _ Measured x-alues partly from Fig_ 5_

311

SiggeI

312

simple possibilities are expected for the half-times after a short For diRerent chloroplast preparations, KTAvaried from I,Z-2,$x TO-~ LJ$_ For long flashes, h”A is somewhat larger, the straight fine being probably an approximation for a more complicated function (Fig. S). _Asin the case of the kinetic equation (I), a choice between the models is not possibie, It is to be hoped that a combined discussion of the dependencies on pH and reduction degree of the pool will favour one of them. Before discussing the problem in the following chapter, some recent measurements of the absorption change at 265 nm shaIi be presented_ After a short fiash the kinetics of ?QE&-oxidation and chl-nr-i--reduction are espected to be equal, regardless of the pH of the medium_ The pattern for the 265 nm absorption change is however more complicated, the time course being biphasic (Fig. gj. The

flash.

Fig. g. Time course of the absorption change at 65 nm after ‘a short fiash for different pH ; gram&din 3X1o-~ ,%I. ferricvanide 5 XECI-5 M_ The half times of the siow phase are indicated.

The pEf--dependence of the slow phase is the same as the one observed for the 703 nm absorption change with a & of 2 x TO-~ 1W (the rate at PR = 6 being too slow, however), With a lower pH the fast phase in-

Plastoquinone

as Electron

and

Proton

Carrier in Photosynthes+

313

creases in magnitude and half-time. It is too early as to give an unequiv~ ocal interpretation. At pH = 7 the 265 nm-signal is reported to consist of a slow exponential phase and of a fast phase of 0,6 ms half-times (not resolved here). The absorption changes. of PQH, and X- are superimposed. It is possible that the absorption change of the plastosemiquinone PQHI interferes at low pH : this may be expected, but has not yet been found.

General discussion of the modeIs The m&we

of PH-control

It has been shown that the o_xidation of PQH,, which gives rise to a proton pump reaction, is retarded by a high internal proton concentration. This has been interpreted as due to the back-pressure of- protons : i.e. back pressure on a redo-x reaction r”?16or only a protonation equilibrium_ l3 The redox reaction is in our view practically not reversible.

Firstly, the equilibrium constant I< is so large that even at a pH=s the reaction progresses completely toward the right side. K=

CPQI

[VI” [Hi+]”

[PQHJ

[U+]’

This follows from the redox potentials of U,, = o Vfor PQ at pH = 7 and of iYO= 0,37 V for plastocyanine. However, even if the:. constant were more favourable, the equilibrium would be shifted to the PQ-side because_ the electrons are constantly withdrawn out of it and therefore not available for a back-reaction. U is rapidly oxidized by light reaction I. Therefore, this type of energetic control has to be rejected in favour of a kinetic control in which a back-pressure is exerted on a proton reaction_ A-+Hi+SAH For the electron reaction to be influenced a relais-mechanism has to be postulated : an electron transfer to the oxidized antipool is only possible after the proton transfer_ This may be due to a conformational change in the. chain, triggered perhaps by the negative charge of P&‘-_

Siggel

3x4

Two possibilities have been proposed for electron as well as for proton translocation in the course of PQH2-oxidation. A total of 1 models results from their intercombination. Two extreme models shall be treated quantitatively here. As a first step the initial oxidation rate at the onset of dark-time (which is equal to the stationary rate of electron transport) will be calculated. Two experimental results WilI be taken as a test for the models ; the rate of oxidation for pH = 65-S (formula I) with k, [P&J = & s-l and the pH-dependence (formnla II) with KA = 3 x IO-~ &I. The fiuid model (A) PQ is able to travel across the membrane, the kinetics is that of a homogeneous solution_ The plastosemiquinone PQH- is responsible for the pH-dependence and releases the protons into the inner phase (Fig. IO bottom). I.

Fig. IO. Schematic representation of the reactions of plastoquinone showing two models of PQH,-osidation with coupled proton Xodel A : Fluid model with deprotonation transiocation. of the semiquinone. Model B I Statistical model with trauslocator molecule A- for the proton.

k,

PQ + PQH, + PQHk-z

k, + PQ*4

+ Hit

k, *

u-t

Plastoquinone

as Electron

and

Proton

Carrier

in photosynthesis.

i

315

:

This model is characterized by the followings differential equations:

..

d PQH-I = z K, [PQHJ ([PQo] L [p&J dt

-

[PQH*12 + k, [Hi+] [PQ;-]

z k,,

--R,

..’

_L [PQH-] -_ XIPQ.$)l 1 [PQHI]

.:

__. &)

_

d

[PQ-] dt

-. k, [Hi+] [PQ=-] L- ks [U+] [PQ’--] :- ; _. (2);

= k, [PQH.] -

The stationary rate of electron transport (2) is given by :.

-__

f = k&G,][PQ--1s The steady state [PQ’--Iss follows directly. from (2,. .\lvhereas[PQH’], from (I) and (2) is the solution of a quadratic equation, ..

tPQ’-1s= [PQH-1s

=

[IPQH%

k,

Iv,l-$;

cHi+l

--

ks ks [Uol + 2 k2 [PQHJs (ks + ks @JoIt 4 k-_2 (ka [Uo + k* ,!X+l).-

+1/r+ (ks ks [.UoJ+ 16&k-+

[PQH&

([PQJ-[PQHis) -

2 k2 [PQH&

(3)

-:

ka CHi~ll

x

(&[Uol fka[Hi+l)2. (KS’t. ks [Uol + h[&+1)12-

:_I .(&j’

Accordingly, the expression for e’ is rather complicated. ’ In order to arrive at the simple experimental equation (I) for high pH; t&o. &$proximations are necessary. The expansion of the square_ root into- a TAYLOR series and breaking off after the -second term yields a. gmkh simpler formula. 1 .

e 5=: 2

-

k, [PQH&

CPQW=)

([PQo] -

s

-’ .

k,K,[U,I

& k3 [Uo] -/- z R, [PQHJxs (hi+&

/ ‘-_

.’

[Uol. + k, [Hi+])

(k, + k, ko]

-’ :

..is,. ~:

..

which corresponds to (I) in case of : k, k, [Ut,-j S 2 k, [PQH&

-- ...

+

kq [Hi+])

: .--

To answer the question, if the complete formula for e’ describes.. the experimental pH-depend&nce (II), it is assumed. that the break-. off. -of the series has to. be valid for all the pH’s used; It is :appropriate, to eeas-semble (5) .and to. define an effective dissociation constant K;i (KS is the. true dissociation constant of the semiquinone). 1 --

316

(7) It is obvious that the simple function for the pH-dependence of i (or I/&) is approximately met by (6) if [PQHJ, does not vary too much with pH. The necessary requirements for the magnitude of the unknown rate constants to yield h-ef = 3 x ro-6 (taking [PQII& = 3) are listed in Table I_

Possible values of k, to yield Kef = 3 x 10:~ M, calculated from Table I_ The rate constants k, and equation (7). 3 values of h; (in Af) bein, - assumedthe condition that breaking off the TAYLOR series must k, are derived from Dimension of k, is 31-l S-I, that of k_, and k, be valid for 5 < PHi < S. sdl mole centers II/mole.

I

I i

j I

K,

’

k -2

k,

I.

k,

i Io-s

Io-2

6x10~

;

i

1010

L4XIO3

1

I010

10-3

g_sxro3

1

103

10-3

=j_sx103

I

i 1 I

j 10-T t

I I

10-z ~~10-3

I

I03 I

i

We conclude that model A is formally possible_ It is characterized by the fact that both the disjcommutation equilibrium PQH,, PQ/PQW (&) and the protonation equilibrium of PQH. [KS} are displaced by successive reactions. The rate limiting reaction is the formation of PQW around pH = 7, whereas the slow rate at pH = 5 is due to the small concentration of the reactive species PQ*-. However, the model does not seem to be a very probable one : -At pH = 5 a large portion of the reducing equivalents in the pool is presThe corresponding equilibrium constant ent as semiquinone PQHI. & = k,/k_, is very high (~~00) compared with values calculated (Ref13) from measurements of simple benzoquinones in aqueous solution (10-y.

Plastoquinone as Electron and. Proton .Carrier in Phbtos@th&is

L,‘-.3.17 '.

z.

The’ statistical

model

~.

.(B)

:

:

:::

I :__: .'. -y_.;

Here the P&molecdes have g _iimiieai&bility‘\$&& j-J-&$&~ The reducible etig is located- &t the’. &it& ‘si4e. a&d of the membrane: accepts protons from the outer phase.. .Oxid+ioti of PQH,:is ‘b&y pos&l~ : on certain reaction sites. The protons are transferred to a p&on;$$risJ~): cator molecule which releases them into ,the- iriner phase, pg&ibly after rotation (Fig. IO top).

PQ

PQH’ -

PQ%

PQH-

k, u-t -A-

The formula for. the stationary electron transport rate- ‘.can be. written directly by multiplying the probabilities. for those ..states .-of.‘& reactants required for the reaction_ _

with

k.” _ 2

-p-

w k, [IJO] '2

[PQo]”

. . _; .~ :

: ..

..

._

... ... :.

..

;

The formula & .is obviously in agreement &th the~~e&&imental.. result (I). This will also be tiue for the-, empirical.kqnat&n -(II)., if -the probability o of the semiquinone state and .the ‘product [PQII& [PQ];;T are not dependent on the proton concentration_ The -latter r.eqrurement ,&i for:? the- e.+rsis more easily met than that for [PQHJ S in model A.-: tence of a true. equilibrium PQE+, PQ/PQH--is postulated.for _aU,..th$pe(as well as the protonation equrlibrium for -A); ‘Thus -the- ~tekhrtuting --.-.: step remains alivtiys the same. It is hoXvever~ t+e;l. by~r,$IG&& Model Bseems to be possible. .. . I: .. j similar to the one noticed in model A. A much higher concentration of semiquinone,:: @an expected: fro& the results in aqueous solution, is .necess&ry on the.-reaction sitei:.j(giVe.g by w) in order to obtain the electron transpol’t-rates, that were. mea&red:. This may be explained by the special nature of the. environment;. ,The overall semiqtinone concentration will still be low, in accordatice, with EPR measurements_ Thus model B seems to be more probable ’ thari model A. .. .-

Conclusion -At this time it is difficult to choose one of the‘ models presented as the one describing unequivocally the mechanism of olridation of pla-. The pH-dependence of the electron transport rate stohydroquinone.

Siggel

318

has been successfully described by model B taking into account the pHdependence of the water splitting reaction for [PQHJ, (Ref_ 17) _ However, in view of the complicated UV-absorption changes, this does not seem to be a sufficient proof- The calculations show, however, how to proceed. The most conspicuous difference between the models is, that the kinetic equation (I) is valid at high pH onlv in model A, while it is valid in the whole pH-range in model B. So dkect examination of the equation is desirable- The corresponding experiments are being performed.

References 1

31. KLITGEXSBERG,

(London)

194,

379

A. MUELLER,

P. SCHMIDT-MEXDE

and

H.T.

\'C'ITT,Nafuye

(1962)

2

H-H_ STIEHL and H.T_ \VITT, 2. Nntzr~fo~sch. Teil B 23, 220 (1968) 3 H-H. STIEHL and H.T. \VITT, Z_ A~at~vfo~sch_ T.&E-B 24, 158s (1969) 4 P_ SCHMIDT-MEXDE and H.T. WITT, 2. Xafztyfoysch. Teil B 23, z& (1965) 5 G U_ SIGGEL, G. REM&R, H-H_ STIEHL and B_ RUMBERG, Biochina. Biophys. Acfu 256, 3~8 (rgp) 6 \V_ HXEHSEL, PYOC. III Id. Congr. of Photosynthesis, Vol. I. p_ 557 (x974) 7 B_ RUJLBERG and U. SIGGEL. _vnfzrv~issenscir~ffe?z 56, 130 . tg6g)

8

16

H. SCHB~DER, EL NIJHLE anC B. R~XEERG, PYOG. II Id_ Cost,ar_ Phofosynthesis BPS_, Vol_ II, p_ gig (1972) \\'_ J~SGE and X1:. AUSLXXDER, Biod.&n. Biophp. AC&Z. 333, 5g (1973) H-T. WITT, Q_ Rev. Biophys. 4, 4 (1971) K. WITT. FEBS Letf. 38, 116 (1973) R_ B~ssnssos and E. J_ LAXD, Biochim_ Biofih~~s- Acfa 325, 175 (1973) U. SIGGEL, Thesis, Technical University Berlin (1974) J_ VATER, Thesis, Technical University Berlin (1971) U_ SIGGEL, G. RE~GER and B. RUMIBERG, Proc. II Int. Congy. Photosynthesis Res_, Vol_ I. p_ 753 (rgp) B. RUMBERG, E. REIXWALD, H. SCHR~DER and U. SIGGEL. PYO~Y. Phofo-

I7

synthesis Res., Vol. III, p. 1373 (1969) U. SIGGEL. Proc. III Int_ CON~Y. of Photosynthesis,

9 x0 l1 12 13 14 15

Vol. I, p_ 645

(1974)