The reductive phase of Rhodobacter sphaeroides cytochrome c oxidase disentangled by CO ligation.

Cytochrome c oxidase (CcO) is a membrane protein of the respiratory chain that catalytically reduces molecular oxygen (O) to water while translocating protons across the membrane. The enzyme hosts two copper and two heme iron moieties (heme a/heme a). The atomic details of the sequential steps that go along with this redox-driven proton translocation are a matter of debate. Particularly for the reductive phase of CcO that precedes oxygen binding experimental data are scarce. Here, we use CcO under anaerobic conditions where carbon monoxide (CO) is bound to heme a which in tandem with Cu forms the binuclear center (BNC). Fourier-transform infrared (FTIR) absorption spectroscopy is combined with electro-chemistry to probe different redox and protonation states populated by variation of the external electrostatic potential. With this approach, the redox behavior of heme a and the BNC could be separated and the corresponding redox potentials were determined. We also infer the protonation of one of the propionate side chains of heme a to correlate with the oxidation of heme a. Experimental changes in the local electric field surrounding CO bound to heme a are determined by their vibrational Stark effect and agree well with electrostatic computations. The comparison of experimental and computational results indicates that changes of the heme a/Cu redox state are coupled to proton transfer towards heme a. The latter supports the role of the heme a propionate D as proton loading site.