bioRxiv. 2023 Mar 22. pii: 2023.03.20.530986. [Epub ahead of print]
Izumi Ishigami,
Raymond G Sierra,
Zhen Su,
Ariana Peck,
Cong Wang,
Frederic Poitevin,
Stella Lisova,
Brandon Hayes,
Frank R Moss,
Sébastien Boutet,
Robert E Sublett,
Chun Hong Yoon,
Syun-Ru Yeh,
Denis L Rousseau.
Cytochrome c oxidase (C c O) is an essential enzyme in mitochondrial and bacterial respiration. It catalyzes the four-electron reduction of molecular oxygen to water and harnesses the chemical energy to translocate four protons across biological membranes, thereby establishing the proton gradient required for ATP synthesis 1 . The full turnover of the C c O reaction involves an oxidative phase, in which the reduced enzyme ( R ) is oxidized by molecular oxygen to the metastable oxidized O H state, and a reductive phase, in which O H is reduced back to the R state. During each of the two phases, two protons are translocated across the membranes 2 . However, if O H is allowed to relax to the resting oxidized state ( O ), a redox equivalent to O H , its subsequent reduction to R is incapable of driving proton translocation 2,3 . How the O state structurally differs from O H remains an enigma in modern bioenergetics. Here, with resonance Raman spectroscopy and serial femtosecond X-ray crystallography (SFX) 4 , we show that the heme a 3 iron and Cu B in the active site of the O state, like those in the O H state 5,6 , are coordinated by a hydroxide ion and a water molecule, respectively. However, Y244, a residue covalently linked to one of the three Cu B ligands and critical for the oxygen reduction chemistry, is in the neutral protonated form, which distinguishes O from O H , where Y244 is in the deprotonated tyrosinate form. These structural characteristics of O provide new insights into the proton translocation mechanism of C c O.