A CRUCIAL ROLE FOR ASPL213 IN THE PROTON-TRANSFER PATHWAY TO THE SECONDARY QUINONE OF REACTION CENTERS FROM RHODOBACTER-SPHAEROIDES

The aspartic acid at position 213 of the L-subunit (AspL213) of reaction centers from Rb. sphaeroides is one of two acidic residues in the binding site of the secondary quinone (QB). Alteration of AspL213 to asparagine by site-directed mutagenesis drastically affected the light-induced proton and electron transfer functions leading to formation of quinol (QBH2). The first electron transfer was slowed to a half-time in the millisecond time range, but the equilibrium (Q-AQB ↔ QAQ-B) was substantially increased in favor of QB reduction and the pH dependence of the equilibrium was altered. The stabilization of Q-B is suggested to result from the uncharged nature of the substitution, with the implication that AspL213 is normally ionized and presents an electrostatic restriction to the first electron transfer. The second electron transfer (Q-AQ-B + 2H+ ↔ QAQBH2) was even more severely inhibited and was at least 104-times slower than the wild type at pH > 6.5, and after only two flashes the RCs were blocked in the Q-AQ-B state. At lower pH some transfer activity was restored, although with a rate still 103-times slower than the wild type. The kinetics of the second electron transfer at low pH corresponded exactly to the kinetics of proton uptake. These data are interpreted as implying an essential role for AspL213 in the proton transfer pathway leading to the formation of QH2 after the second flash. © 1990.