Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2009 Feb 17;106(7):2165-9.
doi: 10.1073/pnas.0806391106. Epub 2009 Jan 21.

A peroxide bridge between Fe and Cu ions in the O2 reduction site of fully oxidized cytochrome c oxidase could suppress the proton pump

Affiliations

A peroxide bridge between Fe and Cu ions in the O2 reduction site of fully oxidized cytochrome c oxidase could suppress the proton pump

Hiroshi Aoyama et al. Proc Natl Acad Sci U S A. .

Abstract

The fully oxidized form of cytochrome c oxidase, immediately after complete oxidation of the fully reduced form, pumps protons upon each of the initial 2 single-electron reduction steps, whereas protons are not pumped during single-electron reduction of the fully oxidized "as-isolated" form (the fully oxidized form without any reduction/oxidation treatment) [Bloch D, et al. (2004) The catalytic cycle of cytochrome c oxidase is not the sum of its two halves. Proc Natl Acad Sci USA 101:529-533]. For identification of structural differences causing the remarkable functional difference between these 2 distinct fully oxidized forms, the X-ray structure of the fully oxidized as-isolated bovine heart cytochrome c oxidase was determined at 1.95-A resolution by limiting the X-ray dose for each shot and by using many (approximately 400) single crystals. This minimizes the effects of hydrated electrons induced by the X-ray irradiation. The X-ray structure showed a peroxide group bridging the 2 metal sites in the O(2) reduction site (Fe(3+)-O(-)-O(-)-Cu(2+)), in contrast to a ferric hydroxide (Fe(3+)-OH(-)) in the fully oxidized form immediately after complete oxidation from the fully reduced form, as has been revealed by resonance Raman analyses. The peroxide-bridged structure is consistent with the reductive titration results showing that 6 electron equivalents are required for complete reduction of the fully oxidized as-isolated form. The structural difference between the 2 fully oxidized forms suggests that the bound peroxide in the O(2) reduction site suppresses the proton pumping function.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Effects of X-ray irradiation on the visible absorption spectra of the single crystals of the fully oxidized “as isolated” bovine heart cytochrome c oxidase. The measuring light beam focused to ≈50 μm in diameter was injected perpendicularly to the most extended plane of (010). The X-ray beam injected along the plane was thick enough to irradiate the entire area of the crystals that was irradiated with the measuring light beam. (A and B) The visible spectra are shown for the sample before the X-ray irradiation (A) and after a 30-second exposure (B). (C and D) Time courses of increases in absorption difference are shown between 604 and 630 nm (C) and between 582 and 630 nm (D).
Fig. 2.
Fig. 2.
(Fo-Fc) difference electron density maps for the 1- s and 15-s datasets, calculated at 2.5-Å resolution. The datasets are depicted together with the structural model of heme a3, CuB, and 3 water molecules. Heme a3, CuB, and the oxygen atom of each of the water molecules are colored in red, green, and light blue, respectively. Three water molecules, 2,007, 2,014 and 2,039, were not included in the structural refinements performed to compare their electron densities with the electron density between Fea3 and CuB. (A) The cages of the difference map for the 1-s data are drawn at the 4.5σ level. (B) The cages for the 15-s data are drawn at the 5σ level.
Fig. 3.
Fig. 3.
(Fo-Fc) difference electron density map of the 15-s data calculated at 2.1-Å resolution. (A)Electron density cages for the electron density between and Fea3 and CuB, and for 3 water molecules depicted at the 5.2σ level in a stick model around the O2 reduction site. Carbon, nitrogen, and oxygen atoms are colored in yellow, blue, and red, respectively. (B) (Fo-Fc) difference maps for the 15-s dataset obtained under 3 different constraints on the Oformula imageO distance. Cages are depicted at the 3.8σ level. Heme a3 is depicted by a ball-and-stick model. Carbon, nitrogen, and oxygen atoms are yellow, blue, and red, respectively. The peroxide anion is illustrated by a stick model. Fea3 and CuB are represented in brown and green. The map of the 1.6-Å constraint (pink) has residual density peaks at both ends of the Oformula imageO bond, whereas the map of the 1.8-Å constraint (green) has a large residual density at the middle of the Oformula imageO bond. The cages for the 1.7-Å constraint (blue) have the smallest residual density.
Fig. 4.
Fig. 4.
Coordination geometries of the peroxide anion obtained from structural refinement of the 15-s dataset calculated at 2.1-Å resolution. The interatomic distances are given in angstroms. Other distances and angles are Fea3-CuB, 4.87 Å; Nε2(H376)- Fea3-O1, 168.5°; Fea3-O1-O2, 144.1° and O1-O2-CuB, 90.5°. These geometries are fully consistent with those of the X-ray structure determined from the 103.5-s exposures at 1.95-Å resolution.

References

    1. Ferguson-Miller S, Babcock GT. Heme/copper terminal oxidases. Chem Rev. 1996;96:2889–2908. - PubMed
    1. Moody AJ, Cooper CE, Rich PR. Characterisation of ‘fast’ and ‘slow’ forms of bovine heart cytochrome-c oxidase. Biochim Biophys Acta. 1991;1059:189–207. - PubMed
    1. Jones MG, et al. A re-examination of the reactions of cyanide with cytochrome c oxidase. Biochem J. 1984;220:57–66. - PMC - PubMed
    1. Thörnström PE, Nilsson T, Malmström BG. The possible role of the closed–open transition in proton pumping by cytochrome c oxidase: The pH dependence of cyanide inhibition. Biochim Biophys Acta. 1988;935:103–108. - PubMed
    1. Mochizuki M, et al. Quantitative reevaluation of the redox active sites of crystalline bovine heart cytochrome c oxidase. J Biol Chem. 1999;274:33403–33411. - PubMed

Publication types

Associated data