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 107 M-1 s-1 and 104, 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+ O2 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 O2 reduction.
Bibliographical noteFunding Information:
This research is sponsored by Department of Science and Technology, India (SB/S1/IC-25/2013) and U.S. National Institute of Health (GM062211).
© 2018 American Chemical Society.
- O reduction reaction
- biosynthetic model
- cytochrome c oxidase
- kinetic isotope effect
- kinetic rate constant
- proton transfer residues