An elementary reaction step of the proton pump is revealed by mutation of tryptophan-164 to phenylalanine in cytochrome c oxidase from Paracoccus denitrificans.
Cytochrome c oxidase couples reduction of dioxygen to water to translocation of protons over the inner mitochondrial or bacterial membrane. A likely proton acceptor for pumped protons is the Delta-propionate of heme a(3), which may receive the proton via water molecules from a conserved glutamic acid (E278 in subunit I of the Paracoccus denitrificans enzyme) and which receives a hydrogen bond from a conserved tryptophan, W164. Here, W164 was mutated to phenylalanine (W164F) to further explore the role of the heme a(3) Delta-propionate in proton translocation. FTIR spectroscopy showed changes in vibrations possibly attributable to heme propionates, and the midpoint redox potential of heme a(3) decreased by approximately 50 mV. The reaction of the oxidized W164F enzyme with hydrogen peroxide yielded substantial amounts of the intermediate F' even at high pH, which suggests that the mutation rearranges the local electric field in the binuclear center that controls the peroxide reaction. The steady-state proton translocation stoichiometry of the W164F enzyme dropped to approximately 0.5 H(+)/e(-) in cells and reconstituted proteoliposomes. Time-resolved electrometric measurements showed that when the fully reduced W164F enzyme reacted with O(2), the membrane potential generated in the fast phase of this reaction was far too small to account either for full proton pumping or uptake of a substrate proton from the inside of the proteoliposomes. Time-resolved optical spectroscopy showed that this fast electrometric phase occurred with kinetics corresponding to the transition from state A to P(R), whereas the subsequent transition to the F state was strongly delayed. This is due to a delay of reprotonation of E278 via the D-pathway, which was confirmed by observation of a slowed rate of Cu(A) oxidation and which explains the small amplitude of the fast charge transfer phase. Surprisingly, the W164F mutation thus mimics a weak block of the D-pathway, which is interpreted as an effect on the side chain isomerization of E278. The fast charge translocation may be due to transfer of a proton from E278 to a "pump site" above the heme groups and is likely to occur also in wild-type enzyme, though not distinguished earlier due to the high-amplitude membrane potential formation during the P(R)--> F transition.
Ribacka
Camilla
C
Helsinki Bioenergetics Group, Program for Structural Biology and Biophysics, Institute of Biotechnology, University of Helsinki, PB 65 (Viikinkaari 1), FIN-00014, Helsinki, Finland. camilla.ribacka@helsinki.fi
Verkhovsky
Michael I
MI
Belevich
Ilya
I
Bloch
Dmitry A
DA
Puustinen
Anne
A
Wikström
Mårten
M
D019943:Amino Acid Substitution; D002846:Chromatography, Affinity; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000302:isolation & purification; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D010230:Paracoccus / Q000201:enzymology*; D010649:Phenylalanine / Q000737:chemistry*; D017494:Proton Pumps / Q000737:chemistry*; D013057:Spectrum Analysis / Q000379:methods; D014364:Tryptophan / Q000737:chemistry*
D017494:Proton Pumps; D010649:Phenylalanine; D014364:Tryptophan; D003576:Electron Transport Complex IV