The Deuterium Isotope Effect as a Tool to Investigate Enzyme Catalysis: Proton‐Transfer Control Mechanisms in Cytochrome c Oxidase

Cytochrome c oxidase is a membrane-bound redox-driven proton pump. The coupling of the exergonic electron-transfer reactions from cytochrome c to oxygen to proton translocation across the membrane requires control of internal electron- and proton-transfer reactions. In this work, we focus on the kinetics of electron and proton transfer during those reaction steps that are coupled to proton pumping in cytochrome c oxidase. The results show that during the first pumping step (peroxy oxoferryl transition), proton transfer regulates intramolecular electron transfer. The proton transfer takes place in two steps: (1) Internal proton transfer from a protonatable group, proposed to be Glu(I-286), in the so-called D-pathway, to an oxygen intermediate at the binuclear center (τ ≅ 100 μs); (2) Rapid re-protonation of Glu(I-286) from the bulk solution (τ < 100 μs). Only after proton uptake from solution the last (fourth) electron is transferred “one step closer” towards the binuclear center (from CuA to heme a). During the second proton-pumping step (oxoferryl oxidized), this electron is transferred to the binuclear center, linked to the uptake of a proton through the D-pathway. The electron-transfer rate displays a kinetic-isotope effect (kH/kD) of 6 ± 1 (in a pH range in which the pH dependence of the rate is small), which indicates that the electron transfer is rate-limited by the proton transfer. The entry into the D-pathway (around Asp(I-132)) is composed of a cluster of negatively-charged amino acid residues together with a number of histidines, forming a so-called proton-collecting antenna designed to allow rapid protonation of groups within the proton-transfer pathway.