Regulation Mechanism of Electron Transfer Reaction from Cytochrome C to Cytochrome C Oxidase
Authors: Ishimori Koichiro 
Published: September 2023

The respiratory chain in mitochondria is quite essential for the energy generation in cells, effectively producing “the energy currency in living organisms”, ATP. The driving force for this system is a series of electron transfers form Complex I (NADH:quinone oxidoreductase) to Complex IV (Cytochrome c Oxidase; CcO) in inner mitochondria membrane, and these electron transfer reactions promote the proton pumping from the matrix to the intermembrane space in mitochondria, and, by use of the resulting proton concentration gradient across the membrane, Complex V (ATP synthase) produces ATP. These series of electron transfer reactions terminate at CcO to reduce molecular oxygen to water molecules. The electrons required for this four-electron reduction of molecular oxygen in CcO are donated from a small soluble electron transfer hemoprotein, Cytochrome c (Cyt c), and this electron transfer is also associated with the proton pumping for the ATP generation in the respiratory chain. To examine the molecular regulation mechanism of the electron transfer reaction to terminate the electron transport chain for four-electron reduction of molecular oxygen and proton pumping for the ATP generation, we kinetically followed the electron transfer reaction and analyzed the electron transfer reaction by the Michaelis-Menten equation. By utilizing the Cyt c mutants, we identified the amino acid residues to regulate the electron transfer reactions (Biochem. J., 2020, 477,1565). The kinetic analysis of the electron transfer reaction also revealed that the apparent rate for the electron transfer reaction determined by the steady-state kinetics is much slower than the intramolecular electron transfer rate in the Cyt c – CcO complex. On the other hand, we successfully determined the interaction site between Cyt c and CcO by using NMR (Proc. Natl. Acad. Sci., 2011, 108, 12271) and protein-protein docking simulation (J. Biol. Chem., 2016, 291, 15320). The Cyt c – CcO complex structure estimated from the NMR and protein-protein docking simulation clearly showed that the two redox centers, heme iron in Cyt c and CuA in subunit II of CcO, were located more than 20 Å apart, and the complexes exhibiting shorter redox center distances were energetically unstable. Combined with the results from kinetic and structural studies, we proposed a “conformationally gated electron transfer mechanism”, where the thermal fluctuation of the protein structure controls the electron transfer rate. However, direct experimental evidence that protein conformational fluctuations regulate electron transfer reactions has not been obtained. In this study, we focused on the viscosity of the protein solution as a factor to regulate the conformational fluctuation of the proteins, based on the assumption that high viscosity of the solution suppresses the conformational fluctuation. The electron transfer reaction from Cyt c to CcO was examined in the high viscosity solution and NMR relaxation measurements under high viscosity were also performed to identify the local structural fluctuation responsible for the regulation of the electron transfer rate. Together with the transient complex structures between Cyt c and CcO estimated by the MD simulation, the regulation mechanism of the electron transfer from Cyt c to CcO based on the “conformational gating” will be discussed.