INHIBITOR BINDING TO ISOLATED CHLOROPLAST CYTOCHROME BF COMPLEX

Effects of three inhibitors of quinol oxidation in the chloroplast cyt ochrome bf complex (stigmatellin, tridecylstigmatellin and dibromothym oquinone) were studied in an isolated system comprising Photosystem I (PS I) particles, plastocyanin (PC) and cytochrome bf complex, in the absence of quinol or quinone. Addition of these inhibitors increased t he extent of cytochrome f oxidation after a laser flash created oxidis ed PS I reaction centre (P700) and PC, and decreased somewhat the exte nt of PC oxidation. The re-reduction of oxidised P700 was more complet e than when inhibitor was absent. The data were simulated with reactio ns which included the putative reduction of cytochrome f by the Rieske centre (FeS) and different rate-coefficients according as to whether inhibitor was bound to the bf complex or not. It was concluded that un der the conditions studied the Rieske centre donated electrons to oxid ised cytochrome f and plastocyanin with an average rate coefficient of 35 s(-1). This electron transfer was prevented by any of the three in hibitors, which also increased the equilibrium coefficient for the cyt ochrome f/PC reaction by a maximum factor of two. This increase corres ponded to a decrease in the back reaction coefficient and an increase in the forward rate. The equilibrium coefficient for the reduction of oxidised P700 by PC was about 2 in the absence of inhibitor but increa sed to about 20 in their presence, but only if cytochrome bf complex w as additionally present. This was attributed to the transient formatio n of complexes between P700 with bound plastocyanin, and bf complex. T he operative mid-point potential of FeS, if that of cytochrome f is 37 0 mV, was 390 mV. Deviations in midpoint potentials (P700/plastocyanin ) from solution values were attributed to the bound state of the react ants. Estimates were made of the binding coefficient of each of the th ree inhibitors to p-sites in the cytochrome bf complex in the absence of competing quinol. A stoichiometry of two inhibitors per bf dimer wa s necessary to cause the above changes in reduction potential of cyt f and PC. A result of one inhibitor per dimer was statistically unlikel y, particularly in the case of tridecylstigmatellin.