COMPETITION AMONG PLASTOQUINOL AND ARTIFICIAL QUINONE/QUINOL COUPLES AT THE QUINOL OXIDIZING SITE OF THE CYTOCHROME BF COMPLEX

We have investigated the interaction of plastoquinol (PQH(2)), duroquinol (DQH(2)) and duroquinone (DQ) with the PQH(2) oxidizing site (Q(o) site) of the chloroplast bf complex. In the absence of exogenous quinones or quinols, with an essentially fully reduced PQ pool, the half-time for reduction of cyt b upon a single-turnover actinic flash was approx. 2-2.5 ms, with an initial rate of about 250 s(-1). The same rate was found with an approx. 20% oxidized PQ pool, indicating that this rate is most probably at or near the V-max for the reaction. When the PQ pool was reduced by addition of 100 mu M DQH(2), the rate of cyt b reduction was considerably slower, with a half-time of about 6 ms and an intial rate of about 100 s(-1). Only at much higher concentrations of DQH(2) did the initial rate of cyt b reduction approach that found in samples where PQH(2) was the sole reductant. We concluded that, in the presence of O-2, a fraction of the added DQH(2) was oxidized to DQ which acted as a competitive inhibitor of the Q(o) site. The slowed cyt b reduction kinetics were not observed when the production of DQ was prevented by excluding oxygen from the sample or by pre-reduction of the PQ pool by sodium dithionite. In strictly anaerobic samples titrated with a series of DQ and DQH(2) concentrations, where the PQH(2) pool was at all times essentially completely reduced, we were able to demonstrate a competitive interaction at the Q(o) site among PQH(2), DQH(2) and DQ. By using an appropriate kinetic model consisting of an enzyme (the bf complex), two alternate substrates (PQH(2) and DQH(2)) and one competitive inhibitor (DQ) - we were able to simulate the pre-steady state kinetics of cyt b reduction that resulted from this competition. From these simulations, we concluded that the V-max for oxidation of DQH(2) and PQH(2) were both about 250 s(-1). The predicted binding constants for the species at the Q(o) site depended on the assumed values of the partition coefficients of DQ and DQH(2) into the thylakoid membrane. When estimated partition coefficients for the DQ and DQH(2) were introduced into the simulations, we were able to estimate that the binding constant of PQH(2) to the Q(o) site was greater than or equal to 2 10(4) M(-1). We concluded that, in native thylakoids, with a completely reduced PQ pool, essentially all Q(o) sites were occupied with PQH(2), consistent with a relatively tight binding. Approx. 3 mu M of added DQ is expected to displace PQH, from half of the Q(o) sites. DQH(2) can displace nearly all of the DQ from the sites, but only at high added concentrations. At the high concentrations of DQH(2) typically employed in electron transfer assays typically 0.5-1 mM - nearly all turnovers of the complex occur at the expense of DQH(2). At lower concentrations, in the presence of O-2, competitive inhibition by DQ can severely affect experimental results. We have also found that decyl-ubiquinol (dUQH(2)) is a substrate for the bf complex, but with the product of partition coefficient into the membrane and binding constant into the Q(o) site greater than that of DQH(2) and DQ.