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

Abstract

AbstractCytochrome 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.