title: The fifth electron in the fully reduced caa(3) from Thermus thermophilus is competent in proton pumping.
abstract: The time-resolved kinetics of membrane potential generation coupled to oxidation of the fully reduced (five-electron) caa(3) cytochrome oxidase from Thermus thermophilus by oxygen was studied in a single-turnover regime. In order to calibrate the number of charges that move across the vesicle membrane in the different reaction steps, the reverse electron transfer from heme a(3) to heme a and further to the cytochrome c/Cu(A) has been resolved upon photodissociation of CO from the mixed valence enzyme in the absence of oxygen. The reverse electron transfer from heme a(3) to heme a and further to the cytochrome c/Cu(A) pair is resolved as a single transition with τ~40 μs. In the reaction of the fully reduced cytochrome caa(3) with oxygen, the first electrogenic phase (τ~30 μs) is linked to OO bond cleavage and generation of the P(R) state. The next electrogenic component (τ~50 μs) is associated with the P(R)→F transition and together with the previous reaction step it is coupled to translocation of about two charges across the membrane. The three subsequent electrogenic phases, with time constants of ~0.25 ms, ~1.4 ms and ~4 ms, are linked to the conversion of the binuclear center through the F→O(H)→E(H) transitions, and result in additional transfer of four charges through the membrane dielectric. This indicates that the delivery of the fifth electron from heme c to the binuclear center is coupled to pumping of an additional proton across the membrane.
authors.affiliation: Department of Molecular Energetics of Microorganisms, A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, Russian Federation. siletsky@genebee.msu.su
authors.forename: Sergey A
authors.initials: SA
authors.lastname: Siletsky
authors.forename: Ilya
authors.initials: I
authors.lastname: Belevich
authors.forename: Tewfik
authors.initials: T
authors.lastname: Soulimane
authors.forename: Michael I
authors.initials: MI
authors.lastname: Verkhovsky
authors.forename: Mårten
authors.initials: M
authors.lastname: Wikström
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000378:metabolism*; D007700:Kinetics; D008564:Membrane Potentials; D008954:Models, Biological; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*; D017494:Proton Pumps / Q000378:metabolism*; D013053:Spectrophotometry; D016963:Thermus thermophilus / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D017494:Proton Pumps; D003576:Electron Transport Complex IV; D010100:Oxygen