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Role of copper ion in regulating ligand binding in a myoglobin-based cytochrome C oxidase model.

ABSTRACT: Cytochrome c oxidase (CcO), the terminal enzyme in the mitochondrial respiratory chain, catalyzes the four-electron reduction of dioxygen to water in a binuclear center comprised of a high-spin heme (heme a(3)) and a copper atom (Cu(B)) coordinated by three histidine residues. As a minimum model for CcO, a mutant of sperm whale myoglobin, named Cu(B)Mb, has been engineered, in which a copper atom is held in the distal heme pocket by the native E7 histidine and two nonnative histidine residues. In this work, the role of the copper in regulating ligand binding in Cu(B)Mb was investigated. Resonance Raman studies show that the presence of copper in CO-bound Cu(B)Mb leads to a CcO-like distal heme pocket. Stopped-flow data show that, upon the initiation of the CO binding reaction, the ligand first binds to the Cu(+); it subsequently transfers from Cu(+) to Fe(2+) in an intramolecular process, similar to that reported for CcO. The high CO affinity toward Cu(+) and the slow intramolecular CO transfer rate between Cu(+) and Fe(2+) in the Cu(B)Mb/Cu(+) complex are analogous to those in Thermus thermophilus CcO (TtCcO) but distinct from those in bovine CcO (bCcO). Additional kinetic studies show that, upon photolysis of the NO-bound Cu(B)Mb/Cu(+) complex, the photolyzed ligand transiently binds to Cu(+) and subsequently rebinds to Fe(2+), accounting for the 100% geminate recombination yield, similar to that found in TtCcO. The data demonstrate that the Cu(B)Mb/Cu(+) complex reproduces essential structural and kinetic features of CcO and that the complex is more akin to TtCcO than to bCcO.

SUBMITTER: Lu C

PROVIDER: S-EPMC3761084 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

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Role of copper ion in regulating ligand binding in a myoglobin-based cytochrome C oxidase model.

Lu Changyuan C Zhao Xuan X Lu Yi Y Rousseau Denis L DL Yeh Syun-Ru SR

Journal of the American Chemical Society 20100201 5

Cytochrome c oxidase (CcO), the terminal enzyme in the mitochondrial respiratory chain, catalyzes the four-electron reduction of dioxygen to water in a binuclear center comprised of a high-spin heme (heme a(3)) and a copper atom (Cu(B)) coordinated by three histidine residues. As a minimum model for CcO, a mutant of sperm whale myoglobin, named Cu(B)Mb, has been engineered, in which a copper atom is held in the distal heme pocket by the native E7 histidine and two nonnative histidine residues. I ...[more]

PMID: 20070118

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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.

Dataset Information

Role of copper ion in regulating ligand binding in a myoglobin-based cytochrome C oxidase model.

Publications

Role of copper ion in regulating ligand binding in a myoglobin-based cytochrome C oxidase model.

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