title: Protonation and free energy changes associated with formation of QBH2 in native and Glu-L212-->Gln mutant reaction centers from Rhodobacter sphaeroides.
abstract: Formation of the quinol QBH2 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 QBH2 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- (approximately 1 ms at pH 7.5); the slow phase was approximately 2000-fold slower. The rate constant of the slow phase depended on the redox state of the primary quinone QA; for QA- the rate constant was larger (i.e., 8-fold at pH 6.0) than for QA. The electron and proton transfers to QB- in EQ(L212) RCs were modeled with a two-step scheme as follows: (1) fast, QA-QB- + H+(1)-->QA(QBH)-; (2) slow, QA(QBH)- + H+(2)-->QAQBH2, where reaction 1 involves concomitant electron transfer and proton uptake [Paddock, M. L., McPherson, P. H., Feher, G., & 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 pKa values are shifted by interactions with the charge of (QBH)-. The total steady-state proton uptake stoichiometry was 2.0 H+/2e- at pH < or = 7.5, consistent with the formation of the quinol QBH2 (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 pKa for H+(2) of approximately 8.5 [McPherson, P. H., Okamura, M. Y., & Feher, G. (1993) Biochim. Biophys. Acta 1144, 309-324]. The proton uptake kinetics indicate that Glu-L212 is a 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., & Okamura, M. Y. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6602-6606].(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Physics, University of California, San Diego, La Jolla 92093-0319.
authors.forename: P H
authors.initials: PH
authors.lastname: McPherson
authors.forename: M
authors.initials: M
authors.lastname: Schönfeld
authors.forename: M L
authors.initials: ML
authors.lastname: Paddock
authors.forename: M Y
authors.initials: MY
authors.lastname: Okamura
authors.forename: G
authors.initials: G
authors.lastname: Feher
mesh_terms: D005973:Glutamine / Q000378:metabolism*; D005998:Glycine / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006873:Hydroquinones / Q000378:metabolism*; D007700:Kinetics; D009154:Mutation; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011522:Protons; D012242:Rhodobacter sphaeroides / Q000737:chemistry / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
chemical_list: D006873:Hydroquinones; D045322:Photosynthetic Reaction Center Complex Proteins; D011522:Protons; D005973:Glutamine; D005998:Glycine