Kyoko Shinzawa-Itoh

Structures of metal sites of oxidized bovine heart cytochrome c oxidase at 2.8 A.

The high resolution three-dimensional x-ray structure of the metal sites of bovine heart cytochrome c oxidase is reported. Cytochrome c oxidase is the largest membrane protein yet crystallized and analyzed at atomic resolution. Electron density distribution of the oxidized bovine cytochrome c oxidase at 2.8 A resolution indicates a dinuclear copper center with an unexpected structure similar to a [2Fe-2S]-type iron-sulfur center. Previously predicted zinc and magnesium sites have been located, the former bound by a nuclear encoded subunit on the matrix side of the membrane, and the latter situated between heme a3 and CuA, at the interface of subunits I and II. The O2 binding site contains heme a3 iron and copper atoms (CuB) with an interatomic distance of 4.5 A; there is no detectable bridging ligand between iron and copper atoms in spite of a strong antiferromagnetic coupling between them. A hydrogen bond is present between a hydroxyl group of the hydroxyfarnesylethyl side chain of heme a3 and an OH of a tyrosine. The tyrosine phenol plane is immediately adjacent and perpendicular to an imidazole group bonded to CuB, suggesting a possible role in intramolecular electron transfer or conformational control, the latter of which could induce the redox-coupled proton pumping. A phenyl group located halfway between a pyrrole plane of the heme a3 and an imidazole plane liganded to the other heme (heme a) could also influence electron transfer or conformational control.

Quantitative reevaluation of the redox active sites of crystalline bovine heart cytochrome c oxidase.

Approximately 30% of the iron contained in a bovine heart cytochrome c oxidase preparation was removed by crystallization, giving a molecular extinction coefficient 1.25–1.4 times higher than those reported thus far. Six electron equivalents provided by dithionite were required for complete reduction of the crystalline cytochrome c oxidase preparation. The fully reduced enzyme was oxidized with 4 oxidation equivalents provided by molecular oxygen, giving an absorption spectrum slightly, but significantly, different from that of the original fully oxidized form. Four electron equivalents were required for complete reduction of the O2-oxidized enzyme. The O2-oxidized form, when exposed to excess amounts of O2, was converted to the original oxidized form which required 6 electrons for complete reduction. A slow reduction of the O2-oxidized form without any external reductant added indicates the existence of internal electron donors for heme irons in the enzyme. These results suggest that the 2 extra oxidation equivalents in the original oxidized form, compared with the O2-oxidized form, are due to a bound peroxide produced by O2 and electrons from the internal donors, consistently with a peroxide at the O2 reduction site in the crystal structure of the enzyme (Yoshikawa, S., Shinzawa-Itoh, K., Nakashima, R., Yaono, R., Yamashita, E., Inoue, N., Yao, M., Fei, M. J., Peters Libeu, C., Mizushima, T., Yamaguchi, H., Tomizaki, T., and Tsukihara, T. (1998)Science 280, 1723–1729).

Structural changes of bovine cytochrome c oxidase dependent on the redox states

Structural changes of bovine cytochrome c oxidase dependent on the redox states Kazumasa Muramoto,a,b Hidenori Fujisawa,a Naomine Yano,a Tomoko Maeda,a Kyoko Shinzawa-Itoh,a Eiki Yamashita,b Tomitake Tsukihara,b,c Shinya Yoshikawa,a,b aDepartment of Life Science and bPicobiology Institute, University of Hyogo, (Japan). cInstitute for Protein Research, Osaka University, (Japan). E-mail: muramoto@ sci.u-hyogo.ac.jp

Structural analysis for lipid/protein interactions in bovine heart cytochrome c oxidase

4 inside surface is located at the D-pathway entrance. The zinc binding affinity for the second site suggests that the zinc site is tightly coupled with the proton-pumping site. Recently, we analyzed Zn/Cd-binding to monomeric CcO which gives crystal packing different from that in the dimeric CcO crystal. The x-ray structural analysis showed Znbinding to the Zn2, Zn3 and additional sites including the site near the K-pathway entrance. Several Zn-binding sites have been found on the outside surface. However none of them is located on the subunit I surface from which pumping protons exit.

Proton pump mechanism deduced from high resolution structures of bovine heart cytochrome c oxidase

(2002). A58 (Supplement), C36 PROTON PUMP MECHANISM DEDUCED FROM HIGH RESOLUTION STRUCTURES OF BOVINE HEART Cytochrome C Oxidase K. Muramoto H. Aoyama K. Hirata E. Yamashita T. Akitsu T. Tsukihara K. Shinzawa-Itoh H. Shimada S. Yoshikawa Faculty of Science, Himeji Institute of Technology Faculty of Science, Himeji Institute of Technology Institute for Protein Research, Osaka University School of Medicine, Keiou University

Mechanism of Dioxygen Reduction by Cytochrome c Oxidase as Studied by Time-Resolved Resonance Raman Spectroscopy

Time-resolved resonance Raman (TR3) spectroscopy has been applied to cytochrome c oxidase (CcO) to elucidate the mechanism of dioxygen reduction. Six oxygen isotope-sensitive Raman bands have been identified in the TR3 spectra. The “607-nm species” defined by difference absorption spectrum, which is referenced against the oxidized enzyme, is demonstrated to have an Fe=O heme, although it has long been believed to have an Fe-O-O-X (X=H or CuB) heme. The one-electron reduction of this Fe=O intermediate, which yields the oxoferryl intermediate, is demonstrated to be coupled with proton transfer in the protein. The mechanism of dioxygen reduction by CcO is discussed on the basis of the structures of the reaction intermediates.