Kazumasa Muramoto

Cytochrome c oxidase (CcO) catalyzes oxygen (O2) reduction at the heme a3-CuB site in the transmembrane region of the enzyme. It has been proposed that the hydrophobic channel that connects the transmembrane surface of subunit III through subunit I to the heme a3-CuB site is the O2 transfer pathway. Gas molecules other than O2, including carbon dioxide (CO2) generated in the tricarboxylic acid cycle, should also enter the hydrophobic channel, but it is not clear how these molecules are expelled from CcO. We analyzed the crystal structures of CO2-, nitrous oxide-, and Xe-bound bovine CcO in the oxidized and reduced states at resolutions of 1.75 to 1.85 Å. Binding of Xe in the channel of subunit I near the interface with subunit III supported the proposed O2 transfer pathway. CO2, nitrous oxide, and another Xe were all bound to a common site near the branching point of another hydrophobic channel that branched from the O2 transport channel. Additional Xe atoms were bound in the second channel leading up to the molecular surface on the intermembrane space side, suggesting that under physiological conditions, CO2 that has entered the O2 pathway could be passively expelled through this channel. This channel consists of subunit I and nuclear DNA-coded subunit VIIc, suggesting that the addition of subunit VIIc in the process of molecular evolution of mitochondrial CcO has made the CO2 exhaust pathway.

Cytochrome c oxidase (CcO) reduces O2 in the O2-reduction site by sequential four-electron donations through the low-potential metal sites (CuA and Fea). Redox-coupled X-ray crystal structural changes have been identified at five distinct sites including Asp51, Arg438, Glu198, the hydroxyfarnesyl ethyl group of heme a, and Ser382, respectively. These sites interact with the putative proton-pumping H-pathway. However, the metal sites responsible for each structural change have not been identified, since these changes were detected as structural differences between the fully reduced and fully oxidized CcOs. Thus, the roles of these structural changes in the CcO function are yet to be revealed. X-ray crystal structures of cyanide-bound CcOs under various oxidation states showed that the O2-reduction site controlled only the Ser382-including site, while the low potential metal sites induced the other changes. This finding indicates that these low-potential site-inducible structural changes are triggered by sequential electron-extraction from the low-potential sites by the O2-reduction site and that each structural change is insensitive to the oxidation and ligand-binding states of the O2-reduction site. Because the proton/electron coupling efficiency is constant (1:1), regardless of the reaction progress in the O2-reduction site, the structural changes induced by the low-potential sites are assignable to those critically involved in the proton-pumping, suggesting that the H-pathway, facilitating these low-potential site-inducible structural changes, pumps protons. Furthermore, a cyanide-bound CcO structure suggests that a hypoxia-inducible activator, Higd1a, activates the O2-reduction site without influencing the electron transfer mechanism through the low-potential sites, kinetically confirming that the low-potential sites facilitate proton-pump.

Abstract Antimicrobial resistance (AMR) is a global health problem. Despite the enormous efforts made in the last decade, threats from some species, including drug-resistant Neisseria gonorrhoeae, continue to rise and would become untreatable. The development of antibiotics with a different mechanism of action is seriously required. Here, we identified an allosteric inhibitory site buried inside eukaryotic mitochondrial heme-copper oxidases (HCOs), the essential respiratory enzymes for life. The steric conformation around the binding pocket of HCOs is highly conserved among bacteria and eukaryotes, yet the latter has an extra helix. This structural difference in the conserved allostery enabled us to rationally identify bacterial HCO-specific inhibitors: an antibiotic compound against ceftriaxone-resistant Neisseria gonorrhoeae. Molecular dynamics combined with resonance Raman spectroscopy and stopped-flow spectroscopy revealed an allosteric obstruction in the substrate accessing channel as a mechanism of inhibition. Our approach opens fresh avenues in modulating protein functions and broadens our options to overcome AMR.

Mammalian cytochrome c oxidase (CcO) reduces O2 to water in a bimetallic site including Fea3 and CuB giving intermediate molecules, termed A-, P-, F-, O-, E-, and R-forms. From the P-form on, each reaction step is driven by single-electron donations from cytochrome c coupled with the pumping of a single proton through the H-pathway, a proton-conducting pathway composed of a hydrogen-bond network and a water channel. The proton-gradient formed is utilized for ATP production by F-ATPase. For elucidation of the proton pumping mechanism, crystal structural determination of these intermediate forms is necessary. Here we report X-ray crystallographic analysis at ∼1.8 Å resolution of fully reduced CcO crystals treated with O2 for three different time periods. Our disentanglement of intermediate forms from crystals that were composed of multiple forms determined that these three crystallographic data sets contained ∼45% of the O-form structure, ∼45% of the E-form structure, and ∼20% of an oxymyoglobin-type structure consistent with the A-form, respectively. The O- and E-forms exhibit an unusually long CuB2+-OH- distance and CuB1+-H2O structure keeping Fea33+-OH- state, respectively, suggesting that the O- and E-forms have high electron affinities that cause the O→E and E→R transitions to be essentially irreversible and thus enable tightly coupled proton pumping. The water channel of the H-pathway is closed in the O- and E-forms and partially open in the R-form. These structures, together with those of the recently reported P- and F-forms, indicate that closure of the H-pathway water channel avoids back-leaking of protons for facilitating the effective proton pumping.