THE INTERACTION OF QUINONE AND DETERGENT WITH REACTION CENTERS OF PURPLE BACTERIA .1. SLOW QUINONE EXCHANGE BETWEEN REACTION-CENTER MICELLES AND PURE DETERGENT MICELLES

The kinetics of light-induced electron transfer in reaction centers (R Cs) from the purple photosynthetic bacterium Rhodobacter sphaeroides w ere studied in the presence of the detergent lauryldimethylamine-N-oxi de (LDAO). After the light-induced electron transfer from the primary donor (P) to the acceptor quinone complex, the dark re-reduction of P reflects recombination from the reduced acceptor quinones, Q(A)(-), o r Q(B)(-). The secondary quinone, Q(B), which is loosely bound to the RC, determines the rate of this process. Electron transfer to Q(B) slo ws down the return of the electron to P+, giving rise to a slow phase of the recovery kinetics with time tau(p) approximate to 1 s, whereas charge recombination in RCs lacking Q, generates a fast phase with tim e tau(AP) approximate to 0.1 s. The amount of quinone bound to RC mice lles can be reduced by increasing the detergent concentration. The cha racteristic time of the slow component of P+ dark relaxation, observed at low quinone content per RC micelle (al high detergent concentratio n), is about 1.2-1.5 s, in sharp contrast to expectations from previou s models, according to which the lime of the slow component should app roach the time of the fast component (about 0.1 s) when the quinone co ncentration approaches zero. To account for this large discrepancy, a new quantitative approach has been developed to analyze the kinetics o f electron transfer in isolated RCs with the following key features: 1 ) The exchange of quinone between different micelles (RC and detergent micelles) occurs more slowly than electron transfer from Q(B)(-) to P +; 2) The exchange of quinone between the detergent ''phase'' and the Q(B) binding site within the same RC micelle is much faster than elect ron transfer between Q(A)(-) and P+; 3) The time of the slow component of P+ dark relaxation is determined by (n)(greater than or equal to 1 ), the average number of quinones in RC micelles, calculated only for those RC micelles that have at least one quinone per RC (in excess of Q(A)). An analytical function is derived that relates the time of the slow component of P+ relaxation, tau(p), and the relative amplitude of the slow phase. This provides a useful means of determining the true equilibrium constant of electron transfer between Q(A) and Q(B) (L-AB) , and the association equilibrium constant of quinone binding at the Q (B) site (K-Q(+)). We found that L-AB = 22 +/- 3 and K-Q = 0.6 +/- 0.2 at pH 7.5. The analysis shows that saturation of the Q(B) binding sit e in detergent-solubilized RCs is difficult to achieve with hydrophobi c quinones. This has important implications for the interpretation of apparent dependencies of Q(B) function on environmental parameters (e. g. pH) and on mutational alterations. The model accounts for the effec ts of detergent and quinone concentration on electron transfer in the acceptor quinone complex, and the conclusions are of general significa nce for the study of quinone-binding membrane proteins in detergent so lutions.