THE INTERACTIONS OF DUROQUINOL, DBMIB AND NQNO WITH THE CHLOROPLAST CYTOCHROME-BF COMPLEX

Duroquinol reduces the endogenous plastoquinone pool of chloroplasts only very slowly because of a lack of quinol-quinone transhydrogenase activity of the cytochrome bf complex, but is a fast direct donor into the cytochrome bf complex via the quinol oxidation site. It also accelerates the rate of cytochrome b-563 reoxidation in the presence of NQNO. Results of three independent methods are consistent with a requirement of one DBMIB per bf monomer for full inhibition of bf complex electron transfer activity, demonstrating that a bf complex monomeric unit is capable of electron transfer activity. At low molar ratios, inhibition reverses in the dark but is fully restored after several turnovers of the complex. Inhibition is weakened by high concentrations of duroquinol or decyl-plasto-quinol. Free DBMIB is rapidly reduced to DBMIBH2 by duroquinol. Addition of DBMIBH2 in the dark does not cause a large inhibition of the first turnover of the bf complex. It is proposed that DBMIBH2 does not form a stable inhibitory complex but can be oxidised by the quinol oxidation site of the cytochrome bf complex, but at a rate which is significantly less than that of oxidation of plastoquinol or duroquinol. The tight inhibitory complex involves the semiquinone or quinone form of DBMIB and this bound species is only slowly reduced in the dark back to free DBMIBH2. Subsequent flash oxidation leads to a competition between DBMIBH2, duroquinol and plastoquinol for the quinol oxidation site. NQNO and HQNO are effectors of the quinone reduction site of the chloroplast cytochrome bf complex. In the analogous mitochondrial bc1 complex, HQNO inhibits electron transfer through the quinone reduction site and this inhibition is not simply a result of the modulation of the midpoint potential of the cytochrome b(H) haem. In the cytochrome bf complex, a large increase of the midpoint potentials of the cytochrome b haems has been reported, but we were unable to repeat this observation. Although the extent of oxidant-induced reduction of haems b is increased by N(H)QNO, inhibition of electron transfer through the quinone reduction site is small, especially in the presence of duroquinol, and the electrogenic reaction which is associated with protonmotive function of the enzyme is not impaired even after multiple turnovers. It is proposed that the Q(i) site of the cytochrome bf complex can still turn over sufficiently rapidly in the presence of NQNO or HQNO so that there is little inhibition of turnover, even although transient kinetic behaviour can be markedly affected.