Redox-Dependent Conformational Changes in Cytochrome c Oxidase Suggest a Gating Mechanism for Proton Uptake

Abstract

A role for conformational change in the coupling mechanism of cytochrome c oxidase is the subject of controversy. Relatively small conformational changes have been reported in comparisons of reduced and oxidized crystal structures of bovine oxidase, but none in bacterial oxidases. Comparing the x-ray crystal structures of the reduced (at 2.15 Å resolution) and oxidized forms of cytochrome c oxidase from Rhodobacter sphaeroides, we observe a displacement of heme a 3 involving both the porphyrin ring and the hydroxyl farnesyl tail, accompanied by protein movements in nearby regions, including the mid part of helix VIII of subunit I which harbors key residues of the K proton uptake path, K362 and T359. The conformational changes in the reduced form are reversible upon reoxidation. They result in an opening of the top of the K pathway and more ordered waters being resolved in that region, suggesting an access path for protons into the active site. In all high resolution structures of oxidized Rhodobacter sphaeroides cytochrome c oxidase, a water molecule is observed in the hydrophobic region above the top of the D path, strategically positioned to facilitate the connection of residue E286 of subunit I to the active site or to the proton pumping exit path. In the reduced and reduced plus cyanide structures, this water molecule disappears, implying disruption of proton conduction from the D path under conditions when the K path is open, thus providing a mechanism for alternating access to the active site.Cytochrome c oxidase (CcO) 1 , the terminal enzyme of the electron transfer chain, provides the final electron sink by accepting electrons from reduced cytochrome c and passing them sequentially through Cu A , heme a and finally to its active site, composed of heme a 3 and Cu B . Oxygen binds at this binuclear center, accepts the electrons and is reduced to water. The energy released from oxygen reduction is utilized by CcO to translocate protons from the N (negative)-side of the membrane to the P(positive)-side, to generate an transmembrane proton electrochemical gradient, which is then used for ATP synthesis (for recent reviews, see ref.( 1-3 )). Extensive kinetic and spectroscopic studies have identified intermediate states during the chemical catalysis, yet the details of the oxygen reduction process are not entirely understood. Even more elusive and controversial is the proton pumping mechanism. † This work is supported by National Institutes of Health Grant GM26916 (S. Supporting Information Available: Schematic drawing of the microspectrophotometer used for spectral analysis of single crystal ( Figure S1), comparison of the distances between heme a 3 farnesyl-OH and Y288 I -OH, as well as the distances between Cu B and heme a 3 Fe in different forms of RsCcO (Table SI). This materials is available free of charge via the Internet at http://pubs.acs.org. Two types of protons, termed pump and substrate protons, are taken up from the N-side of the membrane. During each catalytic cycle, four pump protons are...

Crystallographic and online spectral evidence for role of conformational change and conserved water in cytochrome oxidase proton pump

“…The reduction procedure for the crystals was performed in stabilizing solution with 10 mM sodium dithionite at 4°C as previously described (3). After a few minutes of dithionite soaking, the crystals turned green, indicating the reduction of the crystals.…”

“…Spectra of the I-II RsCcO frozen crystals were recorded at 100 K using the online singlecrystal microspectrophotometer in station 14-BM-C, BioCARS, Advanced Photon Source, Argonne National Laboratory (3,35). Annealing was performed on the crystals after X-ray radiation, during which the cooling nitrogen flow was blocked for 3-4 s to allow temperature increase of the crystals.…”

“…(B, Inset) Exchange of chloride with bromide at the original carboxyl site in D132A crystal. The OH form a tight (2.6 Å) hydrogen bond that has been suggested to control the entrance of protons into the active site (3,14,27). Mutations in this pathway cause major loss of enzyme activity, but the remaining activity is still coupled to proton pumping (9,28,29), implying that this pathway is exclusively involved in substrate proton uptake.…”

Crystal structures of nitric oxide reductases provide key insights into functional conversion of respiratory enzymes

“…Extensive study on the proton pumping mechanism in A-type bovine aa 3 COX by Yoshikawa and coworkers indicates the several structural changes around the heme active site and putative proton pumping pathway upon the reduction of the heme and/or the binding of the ligand to the heme (42). Similarly, Ferguson-Miller and co-workers observed the redox change-and/ or ligand binding-induced structural changes in aa 3 COX from Rhodobacter sphaeroides (39). As a transduction pathway for the structural changes, recent time-resolved spectroscopic study on bovine aa 3 COX suggested that the vinyl group of heme a 3 could be involved (48).…”

“…The mechanism of proton transfer and pumping was most extensively studied in A-type COXs. A-type COXs have at least two proton transfer pathways called K-and D-pathways from the inside of the cellular membrane to the active center (39,42). Some protons are used for the catalytic O 2 reduction at the active center, and the other protons are further transferred to the outside of the membrane through proton pumping pathway(s) which is not clearly identified yet.…”