Crystallographic studies of cytochrome c and cytochrome c oxidase.
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ABSTRACT:
SUBMITTER: Tsukihara T
PROVIDER: S-EPMC8826895 | biostudies-literature | 2022 Jan
REPOSITORIES: biostudies-literature
Publications
Crystallographic studies of cytochrome c and cytochrome c oxidase.
Journal of biochemistry 20220101 1
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Project description:Cytochrome c oxidase (CcO), a terminal oxidase in the respiratory chain, catalyzes the reduction of O2 to water coupled with the proton pump across the membrane. Mitochondrial CcO exists in monomeric and dimeric forms, and as a monomer as part of the respiratory supercomplex, although the enzymatic reaction proceeds in the CcO monomer. Recent biochemical and crystallographic studies of monomeric and dimeric CcOs have revealed functional and structural differences among them. In solubilized mitochondrial membrane, the monomeric form is dominant, and a small amount of dimer is observed. The activity of the monomeric CcO is higher than that of the dimer, suggesting that the monomer is the active form. In the structure of monomeric CcO, a hydrogen bond network of water molecules is formed at the entrance of the proton transfer K-pathway, and in dimeric CcO, this network is altered by a cholate molecule binding between monomers. The specific binding of the cholate molecule at the dimer interface suggests that the binding of physiological ligands similar in size or shape to cholate could also trigger dimer formation as a physiological standby form. Because the dimer interface also contains weak interactions of nonspecifically bound lipid molecules, hydrophobic interactions between the transmembrane helices, and a Met-Met interaction between the extramembrane regions, these interactions could support the stabilization of the standby form. Structural analyses also suggest that hydrophobic interactions of cardiolipins bound to the trans-membrane surface of CcO are involved in forming the supercomplex.
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Project description:Comparative analysis of the transcriptional response, as quantified by RNA-Seq, of Mycobacterium tuberculosis (Mtb) during inhibition of the terminal cytochrome oxidases, Cytochrome bd oxidase and Cytochrome bcc:aa3. This study was designed to evaluate the efficacy if a novel small molecule inhibitor of the Cytochrome bd oxidase. Specifically, we compared the transcriptional response of Mtb during inhibition by Q203 with a Cytochrome bd deletion mutant to the transcriptional response of Mtb treated with a combination of Q203 and the purported Cytorchomre bd small molecule inhibitor (ND-011992).
2020-10-07 | GSE159080 | GEO
Project description:Crystal structures in both oxidized and reduced forms are reported for two bacterial cytochrome c oxidase mutants that define the D and K proton paths, showing conformational change in response to reduction and the loss of strategic waters that can account for inhibition of proton transfer. In the oxidized state both mutants of the Rhodobacter sphaeroides enzyme, D132A and K362M, show overall structures similar to wild type, indicating no long-range effects of mutation. In the reduced state, the mutants show an altered conformation similar to that seen in reduced wild type, confirming this reproducible, reversible response to reduction. In the strongly inhibited D132A mutant, positions of residues and waters in the D pathway are unaffected except in the entry region close to the mutation, where a chloride ion replaces the missing carboxyl and a 2-Å shift in N207 results in loss of its associated water. In K362M, the methionine occupies the same position as the original lysine, but K362- and T359-associated waters in the wild-type structure are missing, likely accounting for the severe inhibition. Spectra of oxidized frozen crystals taken during X-ray radiation show metal center reduction, but indicate development of a strained configuration that only relaxes to a native form upon annealing. Resistance of the frozen crystal to structural change clarifies why the oxidized conformation is observable and supports the conclusion that the reduced conformation has functional significance. A mechanism is described that explains the conformational change and the incomplete response of the D-path mutant.
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Project description:Fragment of cytochrome c oxidase subunit I (COI)of insect Raw sequence reads
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Project description:Insect COI gene fragment Genome sequencing and assembly
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Project description:Cytochrome c oxidase (CytcO) is a membrane-bound enzyme, which catalyzes the reduction of di-oxygen to water and uses a major part of the free energy released in this reaction to pump protons across the membrane. In the Rhodobacter sphaeroides aa₃ CytcO all protons that are pumped across the membrane, as well as one half of the protons that are used for O₂ reduction, are transferred through one specific intraprotein proton pathway, which holds a highly conserved Glu286 residue. Key questions that need to be addressed in order to understand the function of CytcO at a molecular level are related to the timing of proton transfers from Glu286 to a "pump site" and the catalytic site, respectively. Here, we have investigated the temperature dependencies of the H/D kinetic-isotope effects of intramolecular proton-transfer reactions in the wild-type CytcO as well as in two structural CytcO variants, one in which proton uptake from solution is delayed and one in which proton pumping is uncoupled from O₂ reduction. These processes were studied for two specific reaction steps linked to transmembrane proton pumping, one that involves only proton transfer (peroxy-ferryl, P→F, transition) and one in which the same sequence of proton transfers is also linked to electron transfer to the catalytic site (ferryl-oxidized, F→O, transition). An analysis of these reactions in the framework of theory indicates that that the simpler, P→F reaction is rate-limited by proton transfer from Glu286 to the catalytic site. When the same proton-transfer events are also linked to electron transfer to the catalytic site (F→O), the proton-transfer reactions might well be gated by a protein structural change, which presumably ensures that the proton-pumping stoichiometry is maintained also in the presence of a transmembrane electrochemical gradient. Furthermore, the present study indicates that a careful analysis of the temperature dependence of the isotope effect should help us in gaining mechanistic insights about CytcO.
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Project description:As with other mitochondrial respiratory chain components, marked clinical and genetic heterogeneity is observed in patients with a cytochrome c oxidase deficiency. This constitutes a considerable diagnostic challenge and raises a number of puzzling questions. So far, pathological mutations have been reported in more than 30 genes, in both mitochondrial and nuclear DNA, affecting either structural subunits of the enzyme or proteins involved in its biogenesis. In this review, we discuss the possible causes of the discrepancy between the spectacular advances made in the identification of the molecular bases of cytochrome oxidase deficiency and the lack of any efficient treatment in diseases resulting from such deficiencies. This brings back many unsolved questions related to the frequent delay of clinical manifestation, variable course and severity, and tissue-involvement often associated with these diseases. In this context, we stress the importance of studying different models of these diseases, but also discuss the limitations encountered in most available disease models. In the future, with the possible exception of replacement therapy using genes, cells or organs, a better understanding of underlying mechanism(s) of these mitochondrial diseases is presumably required to develop efficient therapy.
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Project description:Cytochrome c oxidase (CcO) reduces molecular oxygen (O2) to water, coupled with a proton pump from the N-side to the P-side, by receiving four electrons sequentially from the P-side to the O2-reduction site-including Fea3 and CuB-via the two low potential metal sites; CuA and Fea. The catalytic cycle includes six intermediates as follows, R (Fea3 2+, CuB 1+, Tyr244OH), A (Fea3 2+-O2, CuB 1+, Tyr244OH), Pm (Fea3 4+ = O2-, CuB 2+-OH-, Tyr244O•), F (Fea3 4+ = O2-, CuB 2+-OH-, Tyr244OH), O (Fea3 3+-OH-, CuB 2+-OH-, Tyr244OH), and E (Fea3 3+-OH-, CuB 1+-H2O, Tyr244OH). CcO has three proton conducting pathways, D, K, and H. The D and K pathways connect the N-side surface with the O2-reduction site, while the H-pathway is located across the protein from the N-side to the P-side. The proton pump is driven by electrostatic interactions between the protons to be pumped and the net positive charges created during the O2 reduction. Two different proton pump proposals, each including either the D-pathway or H-pathway as the proton pumping site, were proposed approximately 30 years ago and continue to be under serious debate. In our view, the progress in understanding the reaction mechanism of CcO has been critically rate-limited by the resolution of its X-ray crystallographic structure. The improvement of the resolutions of the oxidized/reduced bovine CcO up to 1.5/1.6 Å resolution in 2016 provided a breakthrough in the understanding of the reaction mechanism of CcO. In this review, experimental studies on the reaction mechanism of CcO before the appearance of the 1.5/1.6 Å resolution X-ray structures are summarized as a background description. Following the summary, we will review the recent (since 2016) experimental findings which have significantly improved our understanding of the reaction mechanism of CcO including: 1) redox coupled structural changes of bovine CcO; 2) X-ray structures of all six intermediates; 3) spectroscopic findings on the intermediate species including the Tyr244 radical in the Pm form, a peroxide-bound form between the A and Pm forms, and Fr, a one-electron reduced F-form; 4) time resolved X-ray structural changes during the photolysis of CO-bound fully reduced CcO using XFEL; 5) a simulation analysis for the Pm→Pr→F transition.
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Project description:Cytochrome c oxidases (Coxs) are the basic energy transducers in the respiratory chain of the majority of aerobic organisms. Coxs studied to date are redox-driven proton-pumping enzymes belonging to one of three subfamilies: A-, B-, and C-type oxidases. The C-type oxidases (cbb3 cytochromes), which are widespread among pathogenic bacteria, are the least understood. In particular, the proton-pumping machinery of these Coxs has not yet been elucidated despite the availability of X-ray structure information. Here, we report the discovery of the first (to our knowledge) sodium-pumping Cox (Scox), a cbb3 cytochrome from the extremely alkaliphilic bacterium Thioalkalivibrio versutus. This finding offers clues to the previously unknown structure of the ion-pumping channel in the C-type Coxs and provides insight into the functional properties of this enzyme.
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Project description:We deleted subunits I (cydA) and II (cydB) of the Mycobacterium tuberculosis cytochrome bd menaquinol oxidase. The resulting ΔcydA and ΔcydAB mutants were hypersusceptible to compounds targeting the mycobacterial bc1 menaquinol-cytochrome c oxidoreductase and exhibited bioenergetic profiles indistinguishable from strains deficient in the ABC-type transporter, CydDC, predicted to be essential for cytochrome bd assembly. These results confirm CydAB and CydDC as potential targets for drugs aimed at inhibiting a terminal respiratory oxidase implicated in pathogenesis.
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