Structural and Functional Characterization of Electron Transfer Complex between Cytochrome C and Cytochrome C Oxidase
Authors: Ishimori Koichiro 
Published: July 2022

The electron transfer (ET) system in the respiratory chain is terminated by the four-electron reduction of molecular oxygen in cytochrome c oxidase, concomitant with the proton pumping from the matrix to the intermembrane space in mitochondria. The last ET reaction in the respiratory chain is mediated by a small ET protein, cytochrome c (Cyt c), and the interprotein ET reaction from Cyt c to CcO plays crucial roles in the four-electron reduction of molecular oxygen and proton pumping essential for the ATP generation in mitochondria. Although the well-defined structures of Cyt c and CcO have been already reported and the structure of the ET complex between Cyt c and CcO is indispensable to examine the regulation mechanism for the ET reaction from Cyt c to CcO, the huge molecular weight of CcO as a membrane-bound protein and large dissociation constant of CcO to Cyt c have been prevented us from the detailed analysis of the ET reaction based on molecular spectroscopies and X-ray crystallography. However, we successfully determined the structure of the ET complex between Cyt c and CcO under steady-state turnover conditions by utilizing protein-protein docking simulation combined with the experimentally determined interaction site for CcO in Cyt c and the Michaelis constants for the ET reaction from various kinds of the Cyt c mutants to CcO. The complex structure determined by the simulation revealed that the ET distance between the redox centers of two proteins, heme iron of Cyt c (Fe
c
) and cupper of the CuA site of CcO (CuA), is nearly 23 Å, which is much longer than that previously suggested. To experimentally confirm the ET distance between two redox centers, the ET rate from Cyt c to CcO in the Cyt c – CcO complex was determined by temperature-dependent flow-flash measurements. In the flow-flash measurements, the solution containing carbon monoxide bound CcO was rapidly mixed with the oxygen-saturated solution, and, after the mixing, the four-electron reduction reaction of molecular oxygen in CcO was initiated by irradiating flash light to the reaction mixture to dissociate carbon monoxide from heme a
3 of CcO and to bind molecular oxygen to the heme a
3 site. Associated with the reduction of molecular oxygen, the successive ET reactions from Cyt c to CcO are induced. The ET rate from Fe
c
of Cyt c to CuA of CcO can be determined by the oxidation rate of Fe
c
of Cyt c. Based on the Marcus equation for the ET reaction, the ET distance estimated from the oxidation rate of Cyt c was less than 19 Å, which is substantially shorter than the Fe
c
–CuA distance in the simulated Cyt c–CcO complex. This finding allows us to speculate that the “ET-active complex” showing the ET rate as detected in the flow-flush experiments would have a different binding orientation of Cyt c on CcO from that in the complex, “energetically stable complex”, determined by the previous docking simulation. To structurally characterize the “ET-active complex”, a protein-protein docking simulation under the structural restraint of the short Fe
c
–CuA distance was applied, and we found a stable complex structure showing that the Fe
c
–CuA distance is ~ 18 Å, corresponding to the experimentally determined ET distance (< 18.6 Å). The orientation of Cyt c on CcO in the newly determined Cyt c – CcO complex was significantly different from that in the “energetically stable complex”, suggesting the “conformational gating” where the thermal motions of the protein induce the conformational changes of the protein from the most energetically stable structure to the less stable but more active structure of the proteins. In the ET reaction from Cyt c to CcO, the “ET-active complex showing fast ET rate and the short ET distance between two redox centers would not be the most energetically stable structure in the Cyt c – CcO complexes and the structural fluctuation induced by the thermal motion would be a trigger to the conformational changes from the “energetically stable complex” to the “ET-active complex”. The much slower oxidation rate of Cyt c by CcO observed for the steady-state kinetics measurements also supports the “conformational gating” mechanism in the ET reaction from Cyt c to CcO, where the rate for the conformational changes from the “energetically stable complex” to the “ET-active complex” would be the rate-determinant step for the ET reaction from Cyt c to CcO.