Conformational Gating in Electron Transfer from Cytochrome c to Cytochrome c Oxidase

Understanding the molecular basis of interprotein electron transfer (ET) is essential for elucidating the mechanisms of bioenergetic processes. In this study, we characterize the ET kinetics between cytochrome c (Cyt c ) and cytochrome c oxidase (C c O) by determining the ET rate constant ( k ET ) within their complex using temperature-dependent flow-flash spectroscopy. The measured k ET was on the order of 10 4 s –1, corresponding to an ET distance of ∼13 Å, as estimated via Marcus theory, significantly shorter than the ∼23 Å distance inferred for the ES complex based on docking simulations using Michaelis constants ( K M ). These results provide strong evidence that ET does not occur within the canonical ES complex but rather within a distinct ET-active complex characterized by a shorter ET distance. Docking simulations further support the existence of this ET-active complex by identifying configurations with restricted ET distances. Importantly, the observed k ET is approximately 80 times faster than the catalytic constant ( k cat ), indicating that ET is not the rate-limiting step in the overall Cyt c –C c O reaction. Given that k cat reflects a millisecond-scale conformational transition from the ES complex to the ET-active complex, it is likely governed by the structural fluctuation of the proteins. These findings support a conformational gating mechanism, wherein thermal fluctuations of protein structure critically regulate ET efficiency. This study advances our understanding of protein–protein ET from Cyt c to C c O by highlighting the role of dynamic structural transitions in modulating the reaction kinetics.