PROTONATION AND FREE-ENERGY CHANGES ASSOCIATED WITH FORMATION OF Q(B)H(2) IN NATIVE AND GLU-L212 -] GLN MUTANT REACTION CENTERS FROM RHODOBACTER-SPHAEROIDES

Formation of the quinol Q(B)H(2) in Glu-L212 --> Gln mutant [EQ(L212)] reaction centers (RCs) from Rhodobacter sphaeroides was investigated by measuring the proton uptake (using dyes), UV absorption changes, and free energy changes associated with the two-electron reduction of QB The advantage of using the EQ(L212) RCs for these studies is that the individual protonation steps can be kinetically resolved and analyzed; conclusions reached regarding the mechanism of formation of Q(B)H(2) are expected to apply also to native RCs. The proton uptake by EQ(L212) RCs was strongly biphasic: the fast phase was essentially concomitant with the second electron transfer to QB(similar to 1 ms at pH 7.5); the slow phase was similar to 2000-fold slower. The rate constant of the slow phase depended on the redox state of the primary quinone Q(A); for Q(A)(-) the rate constant was larger (i.e., 8-fold at pH 6.0) than for Q(A). The electron and proton transfers to Q(B)(-) in EQ(L212) RCs were modeled with a two-step scheme as follows: (1) fast, Q(A)(-)Q(B)(-)+H+(1)-->Q(A)(Q(B)H)(-); (2) slow, Q(A)(Q(B)H)(-)+H+(2),Q(A)Q(B)H(2), where reaction 1 involves concomitant electron transfer and proton uptake [Paddock, M. L., McPherson, P. H., Feher, G., and Okamura, M. Y. (1990)Proc. Natl. Acad. Sci. U.S.A. 87, 6803-6807]. The stoichiometry of the fast proton uptake associated with the two-electron reduction of QB varied from 1.1 to 1.4 H+/2e- at pH 6.5-8.5, consistent with the uptake of H+(1) plus an additional fractional proton uptake due to amino acid residues whose pK alpha, values are shifted by interactions with the charge of(Q(B)H)(-). The total steady-state proton uptake stoichiometry was 2.0 H+/2e- at pH less than or equal to 7.5, consistent with the formation of the quinol Q(B)H(2) (reactions 1 and 2). At pH 8.5, the steady-state proton uptake was 1.6+/-0.1 H+/2e-, which is consistent with an apparent pK(a), for H+(2) of similar to 8.5 [McPherson, P. H., Okamura, M. Y., and Feher, G. (1993) Biochim. Biophys. Acta 1144, 309-324]. The proton uptake kinetics indicate that Glu-L212 isa component of the proton transfer chain for H+(2) that connects reduced QB (buried in the RC protein) to the aqueous solvent as proposed previously Paddock, M. L., Rongey, S. H., Feher, G., and Okamura, M. Y. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6602-6606]. To determine which species protonates slowly in EQ(L212) RCs, i.e., either (Q(B)H)(-) (as shown in reaction 2) or an internal residue (e.g., His-L190) that rapidly transfers a proton to (Q(B)H)(-), we measured the UV difference spectrum associated with the slow proton uptake. The difference spectrum resembles that for the protonation of the quinol anion (QH)(-) in 80% ETOH, supporting the model shown in reaction 2. This means that the second electron transfer to Q(B)(-) can occur with only one proton as shown in reaction 1. The free energy changes Delta G(1) degrees and Delta G(2) degrees; associated with reactions 1 and 2, respectively, were deduced from the equilibrium partitioning.(measured spectroscopically) between Q(A)Q(B)(-) and Q(B)(2-). The free energy Delta G(1) degrees. Is linear with pH between 8.0 and 9.5 with a slope of 63+/-3 meV/pH as expected for the uptake of one [i.e., H+(1)] proton. At pH <9.0, Q(A)(Q(B)H)(-) is energetically favored relative to Q(A)(-)Q(B)(-), while for pH >9.0 Q(A)(-)Q(B)(-) is energetically favored. The free energy Delta G(2) degrees, is >O at pH >9.0 indicating that the pK(a), associated with Hf(2) is <9.0, in agreement with the pK(a) of similar to 8.5 observed for the proton uptake. From the free energy changes one can in principle determine whether the activated intermediate state in reaction 1 is the unprotonated state Q(A)Q(B)(2-) or the protonated state Q(A)(-)(Q(B)H) An analysis using simplifying assumptions favors Q(A)Q(B)(2-) as the intermediate state.