title: Role of the -PEWY-glutamate in catalysis at the Q(o)-site of the Cyt bc(1) complex.
abstract: We re-examine the pH dependence of partial processes of ubihydroquinone (QH(2)) turnover in Glu-295 mutants in Rhodobacter sphaeroides to clarify the mechanistic role. In more crippled mutants, the bell-shaped pH profile of wildtype was replaced by dependence on a single pK at ~8.5 favoring electron transfer. Loss of the pK at 6.5 reflects a change in the rate-limiting step from the first to the second electron transfer. Over the range of pH 6-8, no major pH dependence of formation of the initial reaction complex was seen, and the rates of bypass reactions were similar to the wildtype. Occupancy of the Q(o)-site by semiquinone (SQ) was similar in the wildtype and the Glu→Trp mutant. Since heme b(L) is initially oxidized in the latter, the bifurcated reaction can still occur, allowing estimation of an empirical rate constant <10(3)s(-1) for reduction of heme b(L) by SQ from the domain distal from heme b(L), a value 1000-fold smaller than that expected from distance. If the pK ~8.5 in mutant strains is due to deprotonation of the neutral semiquinone, with Q(•-) as electron donor to heme b(L), then in wildtype this low value would preclude mechanisms for normal flux in which semiquinone is constrained to this domain. A kinetic model in which Glu-295 catalyzes H(+) transfer from QH•, and delivery of the H(+) to exit channel(s) by rotational displacement, and facilitates rapid electron transfer from SQ to heme b(L) by allowing Q(•-) to move closer to the heme, accounts well for the observations.
authors.affiliation: Department of Biochemistry, University of Illinois, Urbana, IL 61801, USA.
authors.forename: Doreen
authors.initials: D
authors.lastname: Victoria
authors.forename: Rodney
authors.initials: R
authors.lastname: Burton
authors.forename: Antony R
authors.initials: AR
authors.lastname: Crofts
mesh_terms: D000968:Antimycin A / Q000031:analogs & derivatives / Q000494:pharmacology; D055162:Biocatalysis; D014450:Electron Transport Complex III / Q000737:chemistry* / Q000378:metabolism; D018698:Glutamic Acid / Q000502:physiology*; D006418:Heme / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006873:Hydroquinones / Q000737:chemistry; D010084:Oxidation-Reduction; D011522:Protons
chemical_list: D006873:Hydroquinones; D011522:Protons; C032456:antimycin; D018698:Glutamic Acid; D006418:Heme; D000968:Antimycin A; D014450:Electron Transport Complex III