Redox induced protonation of heme propionates in cytochrome c oxidase: Insights from surface enhanced resonance Raman spectroscopy and QM/MM calculations

Abstract

Understanding the coupling between heme reduction and proton translocation in cytochrome c oxidase (CcO) is still an open problem. The propionic acids of heme a(3) have 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 CH2 twisting modes of the propionates to the protonation state of the individual heme a and heme a(3) propionates respectively. The comparison between calculated and measured H2O-D2O 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 a(3) only protonation of one heme a(3) propionates was observed. Our data supports the postulated scenario where the heme a(3) propionates are involved in the proton pathway. (C) 2016 Elsevier B.V. All rights reserved.