A MODEL OF THE TWO ELECTRON GATE IN CHLOROPLASTS


       Oxidation of QA- leading to re-opening of photosystem II can be followed by the decline in chlorophyll fluorescence yield after a brief actinic flash. The kinetics after a single flash from the dark-adapted state reflect the electron transfer to an oxidized plastoquinone, QB, to form QB- (the semiquinone) at the QB-site. Kinetic models for the reactions of the two-electron gate in bacterial reaction centers and chloroplasts have been previously proposed, and we have suggested a similar model for green plants on the basis of experiments with pea thylakoid membranes. In our mechanism, a new feature was introduced which explicitly recognized the presence of a fraction of vacant QB-sites in chloroplasts, since this feature was required to account for the biphasic nature of the electron transfer following a single flash from the dark-adapted state. In addition, we were able to account for the pH dependence of Kapp, the apparent equilibrium constant for sharing an electron between primary and secondary quinones, by postulating a single residue close to the QB-site whose pK was changed when an electron was passed to QB to form the semiquinone. We have suggested that H252 serves this function, and shown that a mutant of C. reinhardtii with H252 changed to Q has an inhibited QA- oxidation.

       We have shown that with minor modification, this model also accounts well for the kinetics and thermodynamics of inhibitor binding at the QB-site. We use the same model as the basis for our analysis of kinetic and thermodynamic parameters determining the characteristics of electron transfer in the two-electron gate of susceptible and resistant A. hybridis biotypes.


Fig. 1.

       We assume that the biphasic kinetics in the 10 ms range reflect electron transfer in two distinct populations of photosystem II. Reaction centers after long dark adaption have either a secondary quinone bound at the QB-site, the QB state (QAQB) (blue), or a vacant QB-site (QA.vacant) (yellow), as shown in Fig. 1. An actinic flash produces a singly reduced primary quinone, to give the species QA-.vacant (yellow -> green path), or QA-QB (blue -> green path). Both QA-.vacant and QA-QB are closed centers, and give rise to a high fluorescence yield. At 50 Ás after the flash, when reactions on the donor side have reached equilibrium, the high fluorescence yield reflects the electrons remaining on QA-. In centers in the QB state, the electron is transferred to QB in a first-order process (blue -> green path), generating QAQB-, and giving rise to a low fluorescence, with half time of approximately 150 to 300 Ás depending on biotype. Since the electron on QA can be transferred to QB only when a plastoquinone is present at the QB-site, electron transfer in centers initially with a vacant site must first involve a binding of quinone in a second-order process, and then transfer of the electron (yellow -> green path); one therefore expects for this fraction of centers a slower kinetic, reflecting the convolution of the binding and electron transfer reactions. The biphasic decay kinetics following an actinic flash in the range out to 10 ms can be interpreted as reflecting these two populations. Both phases are well fitted by exponential decay curves. In our experience, the more rapid phase accounts for more than half those centers which transfer an electron within 10 ms. Since the quinone pool is oxidized under our conditions, the minor fraction of initially vacant centers will become occupied from a pool of plastoquinone in > 10-fold excess. This pool will therefore undergo a negligible change in concentration during the reaction, and the process will follow pseudo-first-order kinetics.

       At neutral pH, the proton binding reactions follow the green path, and at low pH (below pK1) the pink -> green path. As the pH is raised above pK2, the rate slows, suggesting that under these conditions the reactions follow the gray -> green path.

       In principle, one can calculate the rate constants for electron transfer and binding reactions directly from the kinetic curves under specific initial conditions, using appropriate rate equations. A simpler alternative makes use of the above model, and the following explicit assumptions:

The solution of eigenvalues of the rate equations give us two eqs. (1) and (2). The apparent equilibrium constant, Kapp, for sharing an electron between the two quinones, the on (kVA) and off (kAV) rate constants, and the dissociation constant, Ko, of plastoquinone, are given in eqs. (3) and (4) by their definitions. KE is the equilibrium constant for the electron transfer reaction (see below for further discussion).

where species in square brackets represent fractions of the reaction centers in a particular state (including protonated species) as indicated in the bracket. QA and QA- represent centers with a vacant QB-site, with the oxidized and the reduced QA respectively. Rate constants are as defined in Table 1, except that in these equations, no account is taken of protonation state, and values will therefore be constant only at a defined pH. Variables r1 and r2 are the apparent rate constants which appear in the solution after solving the rate equations, and their values are obtained directly by applying a non-linear least-squares fitting procedure to the kinetic curves, after the latter have been corrected for the non-linear relation between fluorescence yield and concentration of QA-. Ao and Bo are the amplitudes of the slow and fast components, respectively, of the initial biphasic decay kinetics of QA- following an actinic flash, again obtained directly from kinetic analysis of the data.

       The four rate constants kAV, kVA, kAB and kBA can be calculated at any pH through eqs. 1) - 4) from the four measured parameters, e.g. the fast and slow apparent rate constants, the fraction of vacant (or QB-bound) reaction centers (both determined from the corrected decay kinetics by a non-linear least-squares fitting procedure), and the measurement of the apparent equilibrium constant, Kapp. Kapp is measured from the ratio of the back-reactions in the absence (S2+QB-) or presence of DCMU (S2+QA-).

       The variability of values for rate and equilibrium constants with pH, and the relation to the dependence of Kapp on pH, is given by two different effects:

Table 1.

Physico-chemical constants for the reactions of the two electron gate following an actinic flash in native and atrazine resistant Amaranthus hybridus.

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Constant    Reaction        			susceptible	resistant  	notes
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A.  pK values       								(1)
pK1   QAQB (H+)	--> 	QAQB + H+ 		6.2       	6.2 
pK2   QA.vac (H+)	--> 	QA.vac + H+	6.9       	6.2 
pK3   QAQB- (H+)	--> 	QAQB- + H+ 	8.1       	7.0 
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B.  Equilibrium (K) or rate (k) constants.
Ko,pK  QA-QB	--> 	QA- (+ Qpool)		0.12 ▒ 0.02 	0.57 ▒ 0.06 	(2,3)
KE   	QA-QB 	--> 	QAQB-       		3.1 ▒ 0.7      	4.7 ▒ 0.8
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kVA  QA- (+ Qpool)--> 	QA-QB        		500      	650   		(2,4)
k'VA  QA-(H+) (+ Qpool)--> QA-QB(H+)    	500      	650   		(2,4)
kAV  QA-QB	 --> 	QA- (+ Qpool)		 60      	370   		(2,4)
k'AV  QA-QB(H+) --> 	QA-(H+)(+ Qpool)	300     	3,370   	(2,4)
kAB   QA-QB	--> 	QAQB-         		3,000    	10,000   	(5,6)
k'AB   QA-QB(H+) 	--> 	QAQB-(H+)      	3,000    	10,000   	(5,6)        
kBA   QAQB- 	--> 	QA-QB         		987     	2,200   	(5,6)
k'BA    QAQB-(H+) --> 	QA-QB(H+)      		12      	340   		(5,6)
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Notes.    
(1) It is assumed that values for pK1 and pK2, and the fraction of vacant reaction centers in the dark (determined by Ko), are the same after dark adaptation as those following an actinic flash. (2)  Although these reactions involve the quinone pool, constants shown are based on the pseudo-first-order process, with [Qpool] assumed to be 1. 
(3) The true value can be obtained from Ko = K'o / [PQ]pool; we have previously estimated [PQ]pool = 5 mM under oxidizing conditions [32].
(4)  Rate constants kIJ are for reaction at pH above pK2. Rate constants k'IJ are for reaction at pH below pK1. 
(5)  Rate constants kIJ are for reaction at pH above pK3. Rate constants k'IJ are for reaction at pH below pK1. 
(6) The difference between rate constants k and k' does not reflect a change in the rate constant for the electron transfer process (since KE is independent of pH), but a change in concentration of substrate for the backreaction, [QAQB-], associated with pK3.

Model of the QB-site. Stereo view, showing sites of mutations.

Table 2.

Parameters of the two-electron gate in wild type and herbicide-resistant mutants of Chlamydomonas reinhardtii. t½(QA-) is the half time of the back reaction from QA- to S2 in the presence of DCMU. t½(QB-) is the half time of the back reaction from QB- to S2, measured as the rephasing time of the binary oscillations of the two-electron gate. Ko, the dissociation constant for plastoquinone from the QB site, is given without units, as a relative value for comparison between wild type and mutants.


strain 	 t½	 t½  	Ko       Kapp	  KE	  kAB	  kBA	  kAV	  kVA
	(QA-)	(QB-)	(*)
	 (sec)	(sec)				(ms-1)	(ms-1)	(ms-1)	(ms-1)
w. type	2.7	25	0.40	8.3	11.6	4.29	0.37	0.34	0.86
S264A	2.4	20	2.50	7.3	25.6   15.0	0.56	0.60	0.24
G256D	2.7	25	0.67	8.3	13.8	4.66	0.34	0.47	0.70
V219I	2.0	40	0.32   19.0	25.1	6.25	0.25	0.19	0.60
A251V**	3.0	15	2.47   	4.0	13.9	n.d.	n.d.	n.d.	n.d.
F255Y	2.5	25	0.74	9.0	15.7	3.73	0.24	0.84	1.07
L275F	2.7	20	0.46	6.4	 9.36	3.46	0.37	0.39	0.84

* Ko = Ao / Bo = kAV / kVA

** Assuming 2 components to decay.