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Compound C2, a product of the reaction of oxygen and the mixed-valence state of cytochrome oxidase. Optical evidence for a type-I copper.

ABSTRACT: Compound C2 is a product of the reaction of O2 and the mixed-valence state of cytochrome oxidase. The mixed-valence state of membrane-bound cytochrome oxidase is obtained at -24 degrees C, by using either ferricyanide or yeast peroxidase complex ES as oxidants, and the configurations of oxidized haem a and its associated copper (a3+Cua2+) and of reduced haem a3 and its associated copper (ac3+.CO.Cua3+) are obtained. The mixed-valence-state cytochrome oxidase mixed with O2 at -24 degrees C and flash-photolysed at -60 to -100 degrees C reacts with O2 and initially forms an oxy compound (A2) similar to that formed from the fully reduced state (A1). Thereafter the course of the reaction differs from that obtained in the fully reduced state, and absorbance increases are observed at 740--750 nm and 609 nm and a decrease at 444 nm, with no increase in absorbance at 655 nm. One possible attribution of the absorbance increases is to charge-transfer interaction between the iron of haem a3 and the copper associated with haem a3, Cua3(2+), having properties of a type-I 'blue' copper. A possible attribution of the decrease in absorbance at 444 nm is to liganding of a3(2+). A related explanation is that the 609 nm absorbance involves a charge-transfer interaction of both iron and copper as a mixed-valence binuclear complex, Cua3, having properties of a non-blue copper. Intermediates in addition to Compound C2 are not yet identifiable by chemical or spectroscopic tests. The kinetic and equilibrium properties of Compound C2 are described.

SUBMITTER: Chance B

PROVIDER: S-EPMC1186460 | biostudies-other | 1979 Mar

REPOSITORIES: biostudies-other

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Project description:1. The reaction of the partially reduced mixed-valence state of cytochrome c oxidase (a3+CuA2+a3(2+)COCuB+) with O2 was studied by the rapid-reaction technique of flow-flash spectrophotometry at room temperature. Biphasic absorption records are observed in the time range up to 2 ms in both the Soret and visible spectral regions. The fast-phase rate is O2-concentration-dependent and reaches a pseudo-first-order value of 4.5 X 10(4)s-1 at 680 microM-O2 at 20 degrees C. Under the same conditions the second-phase rate is limited at 6.0 X 10(3)s-1. Kinetic difference spectra of the two species in the Soret region are not markedly different in form, whereas in the visible region two spectroscopically different species are clearly distinguished. 4. The first intermediate has a peak at 595 nm and a trough at 605 nm. The form of this spectrum resembles that seen in low-temperature studies and assigned to an O2-bound form of ferrocytochrome a3. This evidence supports a structure for oxycytochrome c oxidase with O2 bound only to cytochrome a3 and not bridged between cytochrome a3 and CuB. The second intermediate has a difference spectrum with a trough at 592 nm and a peak at 610 nm. Again, the form of this spectrum is similar to that observed during the O2 reaction at low temperature and is though to be a result of electron transfer from the oxidase to bound O2. 5. The oxygen profile of the fast phase suggests that a spectroscopically silent species may precede the formation of compound A. These data represent the first spectroscopic distinction, in the physiological temperature range, between O2 binding and electron transfer during the O2 reaction of mammalian cytochrome c oxidase. 6. A mechanism is presented for the O2 reaction of the mixed-valence state of cytochrome c oxidase involving four intermediate species. Electron transfer during this reaction is slow, relative to that seen with the fully reduced enzyme, and probably accounts for the detectability of the oxyferro species under these conditions.

Project description:UnlabelledCopper 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.ImportanceCopper (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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Compound C2, a product of the reaction of oxygen and the mixed-valence state of cytochrome oxidase. Optical evidence for a type-I copper.

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