PROTON AND ELECTRON-TRANSFER IN THE ACCEPTOR QUINONE COMPLEX OF RHODOBACTER-SPHAEROIDES REACTION CENTERS - CHARACTERIZATION OF SITE-DIRECTED MUTANTS OF THE 2 IONIZABLE RESIDUES, GLUL212 AND ASPL213, IN THE QB BINDING-SITE

Proton and electron transfer events in reaction centers (RCs) from Rhodobacter sphaeroides were investigated by site-directed mutagenesis of glutamic acid at position 212 and aspartic acid at 213 in the secondary quinone (Q(B)) binding domain of the L subunit. These residues were mutated singly to the corresponding amides (mutants L212EQ and L213DN) and together to give the double mutant (L212EQ/L213DN). In the double mutant RCs, the rate of electron transfer from the primary (Q(A)) to the secondary (Q(B)) acceptor quinones is fast (tau almost-equal-to 300-mu-s) and is pH independent from pH 5 to 11. The rate of recombination between the oxidized primary donor, P+, and Q(B)- is also pH independent and much slower (tau almost-equal-to 10 s) than in the wild type (Wt), indicating a significant stabilization of the Q(B)- semiquinone. In the double mutant, and in L213DN mutant RCs at low pH, the P+Q(B)- decay is suggested to occur significantly via a direct recombination rather than by repopulating the P+Q(A)- state, as in the Wt. Comparison of the behavior of Wt and the three mutant RC types leads to the following conclusions: the pK of Asp(L213) in the Wt is almost-equal-to 4 for the Q(A)Q(B) state (pK(Q(B))) and almost-equal-to 5 for the Q(A)Q(B)- state (pK(Q(B)-)); for Glu(L212), pK(Q(B)) almost-equal-to 9.5 and pK(Q(B)-) almost-equal-to 11. In L213DN mutant RCs, pK(Q(B)) of Glu(L212) is less-than-or-equal-to 7, indicating that the high pK values of Glu(L212) in the Wt are due largely to electrostatic interaction with the ionized Asp(L213) which contributes a shift of at least 2.5 pH units. Transfer of the second electron and all associated proton uptake to form Q(B)H-2 is drastically inhibited in double mutant and L213DN mutant RCs. At pH greater-than-or-equal-to 8, the rates are at least 10(4)-fold slower than in Wt RCs. In L212EQ mutant RCs the second electron transfer and proton uptake are biphasic. The fast phase of the electron transfer is similar to that of the Wt, but the extent of rapid transfer is pH dependent, revealing the pH dependence of the equilibrium Q(A)-Q(B)- <-> Q(A)Q(B)H-. The estimated limits on the pK values-pK(Q(A)-Q(B)-) less-than-or-equal-to 7.3, pK(A(A)Q(B)2-) greater-than-or-equal-to 10.4-are similar to those derived earlier for Wt RCs [Kleinfeld et al. (1985) Biochim. Biophys. Acta 809, 291-310] and may pertain to the quinone head group, per se. The slow phase of electron transfer in L212EQ mutant RCs is accompanied by uptake of the second proton to the quinol. It is suggested that in Wt RCs the first proton is delivered to Q(B)- or Q(B)2- directly from Asp(L213), and the second is delivered via Asp(L213) and Glu(L212).