ON THE DETERMINATION OF REDOX MIDPOINT POTENTIAL OF THE PRIMARY QUINONE ELECTRON-ACCEPTOR, Q(A), IN PHOTOSYSTEM-II

Redox titrations of Q(A), the first quinone electron acceptor, have be en performed on Photosystem II (PS II) membranes which were either act ive or inactive in terms of oxygen evolution. The redox state of Q(A) was monitored by measuring the chlorophyll fluorescence yield. When ti trations were done at room temperature in the absence of mediators, an E(m) value of approx. -80 mV was obtained for active centres and appr ox. +65 mV for inactive centres. These values confirm earlier reports (Krieger, A. and Weis, E. (1992) Photosynthetica 27, 89-98) in which m easurements were made under comparable conditions. In addition, we fou nd that these E(m) values were independent of pH from pH 5.5 to pH 7.5 , the range of pH over which the O-2-evolving enzyme is stable. To und erstand better the scattered values for the E(m) of Q(A) which exist i n the literature and to assess the validity of the present values, exp eriments were performed under a range of different titration condition s. Two main experimental factors were found to have a strong influence on the measured E(m) of Q(A). First, the presence of redox mediators at low ambient potentials led to an irreversible shift from the low-po tential (active) form to the high-potential (inactive) form. This is a ttributed to the reduction of the Mn cluster which is thought to remai n out of equilibrium when titrations are done without mediators. Secon dly, upon freezing of samples poised at low potential a change in the redox state of Q(A) occurred, as measured by EPR and fluorescence at l ow temperature. Freezing and thawing of active PS II at potentials whe re Q(A) is chemically reduced results in an irreversible change in the E(m) of Q(A) from the low-potential to the high-potential form. This is accompanied by inhibition of oxygen evolution. It is suggested that this effect might also be related to the reduction of the Mn cluster which is, in this case, induced by freeze-thawing in the presence of c hemically reduced Q(A)(-). Based on these observations, it is suggeste d that most titrations of Q(A) in active PS II that have been reported previously suffer from one or both of these unexpected technical diff iculties, Thus, the E(m) values obtained at room temperature and witho ut mediators are probably those which should be taken into account in understanding the energetics of PS II.