O2 Reduction by Biosynthetic Models of Cytochrome c Oxidase: Insights into Role of Proton Transfer Residues from Perturbed Active Sites Models of CcO

Myoglobin based biosynthetic models of perturbed cytochrome c oxidase (CcO) active site are reconstituted, in situ, on electrodes where glutamate residues are systematically introduced in the distal site of the heme/Cu active site instead of a tyrosine residue. These biochemical electrodes show efficient 4e(-)/4H(+) reduction with turnover rates and numbers more than 10(7) M-1 s(-1) and 10(4), respectively. The H2O/D2O isotope effects of these series of crystallographically characterized mutants bearing zero, one, and two glutamate residues near the heme Cu active site of these perturbed CcO mimics are 16, 4, and 2, respectively. In situ SERRS-RDE data indicate complete change in the rate-determining step as proton transfer residues are introduced near the active site. The high selectivity for 4e(-)/4H(+) O-2 reduction and systematic variation of KSIE demonstrate the dominant role of proton transfer residues on the isotope effect on rate and rate-determining step of O-2 reduction.