Cytochrome c oxidase (CcO), the terminal enzyme of the mitochondrial respiratory chain, catalyzes the four-electron reduction of molecular oxygen to water by accepting electrons from cytochrome c (Cyt c), a single electron carrier. This reaction not only concludes the electron transfer (ET) cascade but also drives ATP synthesis through active proton translocation from the mitochondrial matrix to the intermembrane space. Given the disparity between the single-ET capability of Cyt c and the multi-ET reduction process in CcO, precise regulatory mechanisms are hypothesized to govern this ET reaction. In this study, “flow-flash” measurements were employed to determine the intramolecular ET rate (kET) within the ET complex formed between Cyt c and CcO. The measured kET (~104 s-1) was over 100 times faster than the turnover rate (kcat: ~102 s-1) from the Michaelis-Menten analysis, indicating that the intramolecular ET within the ET complex is not the rate-limiting step for the interprotein ET from Cyt c to CcO. Furthermore, using Marcus theory, the kET was found to exceed by more than 104-fold the estimated ET rate (~1 s-1) predicted for the simulated ES complex structure under turnover conditions, derived from Michaelis-Menten analysis of the ET reaction and NMR-based investigation of interaction sites between Cyt c and CcO. These findings suggest that the ET from Cyt c to CcO proceeds via an “ET-active complex” characterized by a shorter ET distance, rather than through the ES complex formed during the ET reaction. Considering that the interprotein ET reaction from Cyt c to CcO occurs on the millisecond timescale, consistent with tertiary conformational transitions between protein conformations, we propose that the kcat reflects the rate of the conformational transition from the ES complex to the “ET-active complex”. This conformational transition likely represents the rate-limiting step of the interprotein ET from Cyt c to CcO. These results highlight the pivotal role of protein conformational dynamics in regulating the ET reaction, revealing that the ET from Cyt c to CcO is governed by a “conformational gating” mechanism.