The Mg2+-containing Water Cluster of Mammalian Cytochrome c Oxidase Collects Four Pumping Proton Equivalents in Each Catalytic Cycle

Abstract

Bovine heart cytochrome c oxidase (CcO) pumps four proton equivalents per catalytic cycle through the H-pathway, a protonconducting pathway, which includes a hydrogen bond network and a water channel operating in tandem. Protons are transferred by H 3 O ؉ through the water channel from the N-side into the hydrogen bond network, where they are pumped to the P-side by electrostatic repulsion between protons and net positive charges created at heme a as a result of electron donation to O 2 bound to heme a 3 . To block backward proton movement, the water channel remains closed after O 2 binding until the sequential four-proton pumping process is complete. Thus, the hydrogen bond network must collect four proton equivalents before O 2 binding. However, a region with the capacity to accept four proton equivalents was not discernable in the x-ray structures of the hydrogen bond network. The present x-ray structures of oxidized/reduced bovine CcO are improved from 1.8/1.9 to 1.5/1.6 Å resolution, increasing the structural information by 1.7/1.6 times and revealing that a large water cluster, which includes a Mg 2؉ ion, is linked to the H-pathway. The cluster contains enough proton acceptor groups to retain four proton equivalents. The redox-coupled x-ray structural changes in Glu 198 , which bridges the Mg 2؉ and Cu A (the initial electron acceptor from cytochrome c) sites, suggest that the Cu A -Glu 198 -Mg 2؉ system drives redox-coupled transfer of protons pooled in the water cluster to the H-pathway. Thus, these x-ray structures indicate that the Mg 2؉ -containing water cluster is the crucial structural element providing the effective proton pumping in bovine CcO.Cytochrome c oxidase (CcO) 3 reduces molecular oxygen (O 2 ) in a reaction coupled with a proton pumping process. After binding of O 2 to the O 2 reduction site (which includes two redox-active metal sites, heme a 3 and Cu B ), four electron equivalents are sequentially donated from cytochrome c via two additional redox active metal sites, Cu A and heme a. Each of the four electron transfers is coupled to the pumping of a single proton equivalent (1, 2).High resolution x-ray structural studies together with mutational analyses for bovine CcO show a possible proton pumping pathway, known as the H-pathway, which includes a hydrogen bond network and a water channel functioning in tandem (1, 2). The water channel provides access of water molecules (or H 3 O ϩ ions) inside the mitochondrial inner membrane (the N-side) to one end of the hydrogen bond network that extends to the outside of the mitochondrial inner membrane (the P-side). The hydrogen bond network interacts tightly with heme a by forming two hydrogen bonds between the formyl group of heme a and Arg 38 in the hydrogen bond network and between the A-ring propionate of heme a and a fixed water molecule in the hydrogen bond network (1, 2). The net positive charge increase in heme a, which occurs upon electron donation from heme a to the O 2 reduction site to be delocalized to the formyl and propionate gro...

Structure of bovine cytochromecoxidase in the ligand-free reduced state at neutral pH

“…The unit-cell parameters were very similar to those of the oxidized crystal, with deviations of $0.1%, which are comparable to those for crystals obtained at pH 5.7 (Yano et al, 2016). This observation indicates that the CcO molecules in the oxidized and the reduced crystals were very similar in terms of their structures, which affect the molecular packing within the crystals.…”

“…The same procedures as applied for previous structural analyses of CcO crystals obtained at pH 5.7 (Yano et al, 2016) and oxidized crystals obtained at pH 7.3 (Luo et al, 2017) were used for structural determination and refinement. Initial phase angles for structure factors up to a resolution of 4.0 Å were calculated by the molecular-replacement (MR) method (Rossmann & Blow, 1962) using a fully oxidized structure determined at 1.8 Å resolution (PDB entry 1v54; Tsukihara et al, 2003).…”

“…The proton-pumping mechanism, later named the H-pathway mechanism, was proposed based on a structural alteration in ISSN 2053-230X Asp51 of subunit I upon redox change (Yoshikawa et al, 1998). The mechanism was refined based on high-resolution X-ray structural analyses of the oxidized and reduced states, as well as reaction-intermediate analogues (Tsukihara et al, 2003;Aoyama et al, 2009;Muramoto et al, 2010;Yano et al, 2016;Shimada et al, 2017).…”

Biallelic Mutations in MRPS34 Lead to Instability of the Small Mitoribosomal Subunit and Leigh Syndrome

“…42,47 For mitoribosome subunit mapping, the human mitoribosome structure (PDB: 3J9M) was used. 48 For complexes III and IV, homologous human subunits were mapped to the bovine structures PDB: 1BGY and 5B1A, respectively, 49,50 whereas for complex II homologous human subunits were mapped to those found in the porcine structure (PDB: 1ZOY). 51 …”

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

“…Structural differences in helix-X, in various derivatives of bCcO (28), and in its bacterial analogs (29), have been reported previously (SI Appendix, Table S2). Recently, new high-resolution structures of reduced and oxidized bCcO obtained with synchrotron radiation at 50 K were reported (30). To reduce the effects of radiation damage each frame was taken with a 10-s exposure and the crystals were translated by 100 μ after each round of 10 shots.…”