Crystal structure of CO-bound cytochrome c oxidase determined by serial femtosecond X-ray crystallography at room temperature

Abstract

Cytochrome c oxidase (CcO), the terminal enzyme in the electron transfer chain, translocates protons across the inner mitochondrial membrane by harnessing the free energy generated by the reduction of oxygen to water. Several redox-coupled proton translocation mechanisms have been proposed, but they lack confirmation, in part from the absence of reliable structural information due to radiation damage artifacts caused by the intense synchrotron radiation. Here we report the room temperature, neutral pH (6.8), damage-free structure of bovine CcO (bCcO) in the carbon monoxide (CO)-bound state at a resolution of 2.3 Å, obtained by serial femtosecond X-ray crystallography (SFX) with an X-ray free electron laser. As a comparison, an equivalent structure was obtained at a resolution of 1.95 Å, from data collected at a synchrotron light source. In the SFX structure, the CO is coordinated to the heme a 3 iron atom, with a bent Fe-C-O angle of ∼142°. In contrast, in the synchrotron structure, the Fe-CO bond is cleaved; CO relocates to a new site near Cu B , which, in turn, moves closer to the heme a 3 iron by ∼0.38 Å. Structural comparison reveals that ligand binding to the heme a 3 iron in the SFX structure is associated with an allosteric structural transition, involving partial unwinding of the helix-X between heme a and a 3 , thereby establishing a communication linkage between the two heme groups, setting the stage for proton translocation during the ensuing redox chemistry.bioenergetics | X-ray free electron laser | crystallography | cytochrome c oxidase | serial femtosecond crystallography

Insights into functions of the H channel of cytochrome c oxidase from atomistic molecular dynamics simulations

“…Subsequent binding of oxygen closes this channel, and one proton is proposed to be released into the P phase with each reduction/oxidation of heme a via an amide bond gate between Tyr440 and Ser441 (24) and an H-bonded network to Asp51 at the P-phase surface (10). Support has come from structural perturbations induced by redox/ligand state changes in the water channel (25)(26)(27)(28), the Asp51 residue (29), and the proposed proton-collecting site around the bound Mg 2+ (23). These observations, together with effects of H-channel mutations on coupling efficiencies in a chimeric bovine/human CcO construct (30,31), have led to the proposal that these structures provide the route for translocated protons in mammalian mitochondrial CcOs.…”

Radiation chemists look at damage in redox proteins induced by X‐rays

“…The problem of radiation damage is eliminated as data are acquired from each microcrystal before damage occurs with all but the highest intensity sources . The method is still far from routine, but it is starting to be applied to enzyme structures including metalloenzymes, and can be used at room temperature, as demonstrated by the recent determination of a structure of cytochrome c oxidase …”

“…80 The method is still far from routine, but it is starting to be applied to enzyme structures including metalloenzymes, 81 and can be used at room temperature, as demonstrated by the recent determination of a structure of cytochrome c oxidase. 82 ORCID Scot Wherland http://orcid.org/0000-0003-1699-5594…”

A common coupling mechanism for A-type heme-copper oxidases from bacteria to mitochondria

“…Normal electron transfer rates have been reported for all three variants, as would be expected for ideally uncoupled oxidases. These data, together with redox-and ligand-induced structural changes in two domains of the H-channel (56)(57)(58)(59), have led to the proposal that it is the H-channel that provides the route for pumped protons both into and out of the proton trap. However, mutagenesis of H-channel residues of bacterial HCOs failed to support any crucial H-channel function.…”