Redox induced protonation of heme propionates in cytochrome c oxidase: Insights from surface enhanced resonance Raman spectroscopy and QM/MM calculations.
Understanding the coupling between heme reduction and proton translocation in cytochrome c oxidase (CcO) is still an open problem. The propionic acids of heme a3have been proposed to act as a proton loading site (PLS) in the proton pumping pathway, yet this proposal could not be verified by experimental data so far. We have set up an experiment where the redox states of the two hemes in CcO can be controlled via external electrical potential. Surface enhanced resonance Raman (SERR) spectroscopy was applied to simultaneously monitor the redox state of the hemes and the protonation state of the heme propionates. Simulated spectra based on QM/MM calculations were used to assign the resonant enhanced CH2twisting modes of the propionates to the protonation state of the individual heme a and heme a3propionates respectively. The comparison between calculated and measured H2OD2O difference spectra allowed a sound band assignment. In the fully reduced enzyme at least three of the four heme propionates were found to be protonated whereas in the presence of a reduced heme a and an oxidized heme a3only protonation of one heme a3propionates was observed. Our data supports the postulated scenario where the heme a3propionates are involved in the proton pathway.
Institut für Chemie PC 14, Technische Universität Berlin, Strasse des 17. Juni 135, 10623 Berlin, Germany.
Murat
M
Sezer
Institut für Chemie und Biochemie, Freie Universität Berlin, Fabeckstraße 36a, 14195 Berlin, Germany.
Anna-Lena
AL
Woelke
Institut für Chemie und Biochemie, Freie Universität Berlin, Fabeckstraße 36a, 14195 Berlin, Germany.
Ernst Walter
EW
Knapp
Institut für Experimentalphysik, Freie Universität Berlin, Arnimallee 14, 14195 Berlin, Germany.
Ramona
R
Schlesinger
Institut für Chemie PC 14, Technische Universität Berlin, Strasse des 17. Juni 135, 10623 Berlin, Germany.
Maria Andrea
MA
Mroginski
Institut für Chemie PC 14, Technische Universität Berlin, Strasse des 17. Juni 135, 10623 Berlin, Germany.
Inez M
IM
Weidinger
D003576:Electron Transport Complex IV / Q000378:metabolism*; D006418:Heme / Q000378:metabolism*; D010084:Oxidation-Reduction; D011422:Propionates / Q000378:metabolism*; D017494:Proton Pumps / Q000378:metabolism; D011522:Protons; D013059:Spectrum Analysis, Raman / Q000379:methods
D011422:Propionates; D017494:Proton Pumps; D011522:Protons; D006418:Heme; D003576:Electron Transport Complex IV
Cytochrome c oxidase; Proton loading site; Proton pumping; QM/MM calculations; Surface enhanced Raman spectroscopy