Reaction center and UQH2:cyt c2 oxidoreductase act as independent enzymes in Rps. sphaeroides.

Turnover of the ubiquinol oxidizing site of the UQH2:cyt c2 oxidoreductase (b/c1 complex) ofRps. sphaeroides can be assayed by measuring the rate of reduction of cytb561 in the presence of antimycin (AA). Oxidation of ubiquinol is a second-order process, with a value ofk2 of about 3 × 105 M−1. The reaction shows saturation at high quinol concentrations, with an apparentKm of about 6–8 mM (with respect to the concentration of quinol in the membrane). When the quinone pool is oxidized before illumination, reduction of the complex shows a substantial lag (about 1 ms) after a flash, indicating that the quinol produced as a result of the photochemical reactions is not immediately available to the complex. We have suggested that the lag may be due to several factors, including the leaving time of the quinol from the reaction center, the diffusion time to the complex, and the time for the head group to cross the membrane. We have suggested aminimal value for the diffusion coefficient of ubiquinone in the membrane (assuming that the lag is due entirely to diffusion) of about 10−9 cm−2 sec−1. The lag is reduced to about 100 µsec when the pool is significantly reduced, showing that quinol from the pool is more rapidly available to the complex than that from the reaction center. With the pool oxidized, similar kinetics are seen when the reduction of cytb561 occurs through the AA-sensitive site (with reactions at the quinol oxidizing site blocked by myxothiazol). These results show that there is no preferential reaction pathway for transfer of reducing equivalents from reaction center tob/c1 complex. Oxidation of cytb561 through the AA-sensitive site can be assayed from the slow phase of the carotenoid electrochromic change, and by comparison with the kinetics of cytb561. As long as the quinone pool is significantly oxidized, the reaction is not rate-determining for the electrogenic process. On reduction of the pool below 1 quinone per complex, a slowing of the electrogenic process occurs, which could reflect a dependence on the concentration of quinone. If the process is second-order, the rate constant must be about 2–5 times greater than that for quinol oxidation, since the effect on rate is relatively small compared with the effect seen at the quinol oxidizing site when the quinol concentration is changed over theEh range where the first few quinols are produced on reductive titration. When the quinone pool is extracted (experiments in collaboration with G. Venturoli and B. A. Melandri), the slowing of the electrochromic change on reduction of the pool is not enhanced; we assume that this is due to the fact that a minimum of one quinone per active complex is produced by turnover of the quinol oxidizing site. Two lines of research lead us to revise our previous estimate for the minimal value of the quinone diffusion coefficient. These relate to the relation between the diffusion coefficient and the rate constants for processes involving the quinones: (a) The estimated rate constant for reaction of quinone at the AA-site approaches the calculated diffusion limited rate constant, implying an improbably efficient reaction. (b) From a preliminary set of experiments, the activation energy determined by measuring the variation of the rate constant for quinol oxidation with temperature, is about 8 kcal mol−1. Although we do not know the contribution of entropic terms to the pre-exponential factor, the result is consistent with a considerably larger value for the diffusion coefficient than that previously suggested.