X-ray structures of catalytic intermediates of cytochrome c oxidase provide insights into its O₂ activation and unidirectional proton-pump mechanisms.

ABSTRACT: Cytochrome c oxidase (CcO) reduces O₂ to water, coupled with a proton-pumping process. The structure of the O₂-reduction site of CcO contains two reducing equivalents, Fe _{a 3} ²⁺ and Cu_B ¹⁺, and suggests that a peroxide-bound state (Fe _{a 3} ³⁺-O^--O^--Cu_B ²⁺) rather than an O₂-bound state (Fe _{a 3} ²⁺-O₂) is the initial catalytic intermediate. Unexpectedly, however, resonance Raman spectroscopy results have shown that the initial intermediate is Fe _{a 3} ²⁺-O₂, whereas Fe _{a 3} ³⁺-O^--O^--Cu_B ²⁺ is undetectable. Based on X-ray structures of static noncatalytic CcO forms and mutation analyses for bovine CcO, a proton-pumping mechanism has been proposed. It involves a proton-conducting pathway (the H-pathway) comprising a tandem hydrogen-bond network and a water channel located between the N- and P-side surfaces. However, a system for unidirectional proton-transport has not been experimentally identified. Here, an essentially identical X-ray structure for the two catalytic intermediates (P and F) of bovine CcO was determined at 1.8 Å resolution. A 1.70 Å Fe-O distance of the ferryl center could best be described as Fe _{a 3} ⁴⁺ = O^2-, not as Fe _{a 3} ⁴⁺-OH^- The distance suggests an ∼800-cm^-1 Raman stretching band. We found an interstitial water molecule that could trigger a rapid proton-coupled electron transfer from tyrosine-OH to the slowly forming Fe _{a 3} ³⁺-O^--O^--Cu_B ²⁺ state, preventing its detection, consistent with the unexpected Raman results. The H-pathway structures of both intermediates indicated that during proton-pumping from the hydrogen-bond network to the P-side, a transmembrane helix closes the water channel connecting the N-side with the hydrogen-bond network, facilitating unidirectional proton-pumping during the P-to-F transition.

SUBMITTER: Shimada A 

PROVIDER: S-EPMC7186171 | biostudies-literature |

REPOSITORIES: biostudies-literature