Resonance Raman spectral analysis of the heme site structure of cytochrome c oxidase with its positive regulator CHCHD2.

Cytochrome c oxidase (CcO) reduces O, pumps protons in the mitochondrial respiratory chain, and is essential for oxygen consumption in the cell. The coiled-coil-helix-coiled-coil-helix domain-containing 2 (CHCHD2; also known as mitochondrial nuclear retrograde regulator 1 [MNRR1], Parkinson's disease 22 [PARK22] and aging-associated gene 10 protein [AAG10]) is a protein that binds to CcO from the intermembrane space and positively regulates the activity of CcO. Despite the importance of CHCHD2 in mitochondrial function, the mechanism of action of CHCHD2 and structural information regarding its binding to CcO remain unknown. Here, we utilized visible resonance Raman spectroscopy to investigate the structural changes around the hemes in CcO in the reduced and CO-bound states upon CHCHD2 binding. We found that CHCHD2 has a significant impact on the structure of CcO in the reduced state. Mapping of the heme peripheries that result in Raman spectral changes in the structure of CcO highlighted helices IX and X near the hemes as sites where CHCHD2 takes action. Part of helix IX is exposed in the intermembrane space, whereas helix X, located between both hemes, may play a key role in proton uptake to a proton-loading site in the reduced state for proton pumping. Taken together, our results suggested that CHCHD2 binds near helix IX and induces a structural change in helix X, accelerating proton uptake.