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Nitric oxide inhibition of respiration involves both competitive (heme) and noncompetitive (copper) binding to cytochrome c oxidase.

ABSTRACT: NO reversibly inhibits mitochondrial respiration via binding to cytochrome c oxidase (CCO). This inhibition has been proposed to be a physiological control mechanism and/or to contribute to pathophysiology. Oxygen reacts with CCO at a heme iron:copper binuclear center (a(3)/Cu(B)). Reports have variously suggested that during inhibition NO can interact with the binuclear center containing zero (fully oxidized), one (singly reduced), and two (fully reduced) additional electrons. It has also been suggested that two NO molecules can interact with the enzyme simultaneously. We used steady-state and kinetic modeling techniques to reevaluate NO inhibition of CCO. At high flux and low oxygen tensions NO interacts predominantly with the fully reduced (ferrous/cuprous) center in competition with oxygen. However, as the oxygen tension is raised (or the consumption rate is decreased) the reaction with the oxidized enzyme becomes increasingly important. There is no requirement for NO to bind to the singly reduced binuclear center. NO interacts with either ferrous heme iron or oxidized copper, but not both simultaneously. The affinity (K(D)) of NO for the oxygen-binding ferrous heme site is 0.2 nM. The noncompetitive interaction with oxidized copper results in oxidation of NO to nitrite and behaves kinetically as if it had an apparent affinity of 28 nM; at low levels of NO, significant binding to copper can occur without appreciable enzyme inhibition. The combination of competitive (heme) and noncompetitive (copper) modes of binding enables NO to interact with mitochondria across the full in vivo dynamic range of oxygen tension and consumption rates.

SUBMITTER: Mason MG

PROVIDER: S-EPMC1334642 | biostudies-other | 2006 Jan

REPOSITORIES: biostudies-other

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Nitric oxide inhibition of respiration involves both competitive (heme) and noncompetitive (copper) binding to cytochrome c oxidase.

Mason Maria G MG Nicholls Peter P Wilson Michael T MT Cooper Christopher E CE

Proceedings of the National Academy of Sciences of the United States of America 20060109 3

NO reversibly inhibits mitochondrial respiration via binding to cytochrome c oxidase (CCO). This inhibition has been proposed to be a physiological control mechanism and/or to contribute to pathophysiology. Oxygen reacts with CCO at a heme iron:copper binuclear center (a(3)/Cu(B)). Reports have variously suggested that during inhibition NO can interact with the binuclear center containing zero (fully oxidized), one (singly reduced), and two (fully reduced) additional electrons. It has also been ...[more]

PMID: 16407136

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Project description:UNLABELLED:Copper is an essential micronutrient used as a metal cofactor by a variety of enzymes, including cytochrome c oxidase (Cox). In all organisms from bacteria to humans, cellular availability and insertion of copper into target proteins are tightly controlled due to its toxicity. The major subunit of Cox contains a copper atom that is required for its catalytic activity. Previously, we identified CcoA (a member of major facilitator superfamily transporters) as a component required for cbb3-type Cox production in the Gram-negative, facultative phototroph Rhodobacter capsulatus. Here, first we demonstrate that CcoA is a cytoplasmic copper importer. Second, we show that bypass suppressors of a ccoA deletion mutant suppress cbb3-Cox deficiency by increasing cellular copper content and sensitivity. Third, we establish that these suppressors are single-base-pair insertion/deletions located in copA, encoding the major P1B-type ATP-dependent copper exporter (CopA) responsible for copper detoxification. A copA deletion alone has no effect on cbb3-Cox biogenesis in an otherwise wild-type background, even though it rescues the cbb3-Cox defect in the absence of CcoA and renders cells sensitive to copper. We conclude that a hitherto unknown functional interplay between the copper importer CcoA and the copper exporter CopA controls intracellular copper homeostasis required for cbb3-Cox production in bacteria like R. capsulatus. IMPORTANCE:Copper (Cu) is an essential micronutrient required for many processes in the cell. It is found as a cofactor for heme-copper containing cytochrome c oxidase enzymes at the terminus of the respiratory chains of aerobic organisms by catalyzing reduction of dioxygen (O2) to water. Defects in the biogenesis and copper insertion into cytochrome c oxidases lead to mitochondrial diseases in humans. This work shows that a previously identified Cu transporter (CcoA) is a Cu importer and illustrates the link between two Cu transporters, the importer CcoA and the exporter CopA, required for Cu homeostasis and Cu trafficking to cytochrome c oxidase in the cell.

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Nitric oxide inhibition of respiration involves both competitive (heme) and noncompetitive (copper) binding to cytochrome c oxidase.

Publications

Nitric oxide inhibition of respiration involves both competitive (heme) and noncompetitive (copper) binding to cytochrome c oxidase.

Similar Datasets

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