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, an d 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(L21 2) RCs was strongly biphasic: the fast phase was essentially concomita nt 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) th an 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 prot on 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 stoichiom etry of the fast proton uptake associated with the two-electron reduct ion 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 t o amino acid residues whose pK alpha, values are shifted by interactio ns 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, consiste nt with the formation of the quinol Q(B)H(2) (reactions 1 and 2). At p H 8.5, the steady-state proton uptake was 1.6+/-0.1 H+/2e-, which is c onsistent with an apparent pK(a), for H+(2) of similar to 8.5 [McPhers on, 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 reduce d QB (buried in the RC protein) to the aqueous solvent as proposed pre viously 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)(-) (a s shown in reaction 2) or an internal residue (e.g., His-L190) that ra pidly transfers a proton to (Q(B)H)(-), we measured the UV difference spectrum associated with the slow proton uptake. The difference spectr um resembles that for the protonation of the quinol anion (QH)(-) in 8 0% 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 D elta G(2) degrees; associated with reactions 1 and 2, respectively, we re deduced from the equilibrium partitioning.(measured spectroscopical ly) between Q(A)Q(B)(-) and Q(B)(2-). The free energy Delta G(1) degre es. Is linear with pH between 8.0 and 9.5 with a slope of 63+/-3 meV/p H 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)(-), wh ile for pH >9.0 Q(A)(-)Q(B)(-) is energetically favored. The free ener gy Delta G(2) degrees, is >O at pH >9.0 indicating that the pK(a), ass ociated 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 c an in principle determine whether the activated intermediate state in reaction 1 is the unprotonated state Q(A)Q(B)(2-) or the protonated st ate Q(A)(-)(Q(B)H) An analysis using simplifying assumptions favors Q( A)Q(B)(2-) as the intermediate state.