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A spectroscopic investigation of a tridentate Cu-complex mimicking the tyrosine-histidine cross-link of cytochrome C oxidase.

ABSTRACT: Heme-copper oxidases have a crucial role in the energy transduction mechanism, catalyzing the reduction of dioxygen to water. The reduction of dioxygen takes place at the binuclear center, which contains heme a3 and CuB. The X-ray crystal structures have revealed that the C6' of tyrosine 244 (bovine heart numbering) is cross-linked to a nitrogen of histidine 240, a ligand to CuB. The role of the cross-linked tyrosine at the active site still remains unclear. In order to provide insight into the function of the cross-linked tyrosine, we have investigated the spectroscopic and electrochemical properties of chemical analogues of the CuB-His-Tyr site. The analogues, a tridentate histidine-phenol cross-linked ether ligand and the corresponding Cu-containing complex, were previously synthesized in our laboratory (White, K.; et al. Chem. Commun. 2007, 3252-3254). Spectrophotometric titrations of the ligand and the Cu-complex indicate a pKa of the phenolic proton of 8.8 and 7.7, respectively. These results are consistent with the cross-linked tyrosine playing a proton delivery role at the cytochrome c oxidase active site. The presence of the phenoxyl radical was investigated at low temperature using electron paramagnetic resonance (EPR) and Fourier transform infrared (FT-IR) difference spectroscopy. UV photolysis of the ligand, without bound copper, generated a narrow g=2.0047 signal, attributed to the phenoxyl radial. EPR spectra recorded before and after UV photolysis of the Cu-complex showed a g=2 signal characteristic of oxidized copper, suggesting that the copper is not spin-coupled to the phenoxyl radical. An EPR signal from the phenoxyl radical was not observed in the Cu-complex, either due to spin relaxation of the two unpaired electrons or to masking of the narrow phenoxyl radical signal by the strong copper contribution. Stable isotope (13C) labeling of the phenol ring (C1') Cu-complex, combined with photoinduced difference FT-IR spectroscopy, revealed bands at 1485 and 1483 cm(-1) in the 12C-minus-13C-isotope-edited spectra of the ligand and Cu-complex, respectively. These bands are attributed to the radical v7a stretching frequency and are shifted to 1468 and 1472 cm(-1), respectively, with 13C1' labeling. These results show that a radical is generated in both the ligand and the Cu-complex and support the unambiguous assignment of a vibrational band to the phenoxyl radical v7a stretching mode. These data are discussed with respect to a possible role of the cross-linked tyrosine radical in cytochrome c oxidase.

SUBMITTER: Offenbacher A

PROVIDER: S-EPMC2763437 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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A spectroscopic investigation of a tridentate Cu-complex mimicking the tyrosine-histidine cross-link of cytochrome C oxidase.

Offenbacher Adam A White Kimberly N KN Sen Indranil I Oliver Allen G AG Konopelski Joseph P JP Barry Bridgette A BA Einarsdóttir Olöf O

The journal of physical chemistry. B 20090501 20

Heme-copper oxidases have a crucial role in the energy transduction mechanism, catalyzing the reduction of dioxygen to water. The reduction of dioxygen takes place at the binuclear center, which contains heme a3 and CuB. The X-ray crystal structures have revealed that the C6' of tyrosine 244 (bovine heart numbering) is cross-linked to a nitrogen of histidine 240, a ligand to CuB. The role of the cross-linked tyrosine at the active site still remains unclear. In order to provide insight into the ...[more]

PMID: 19438285

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Project description:CuA is a dinuclear mixed-valence center located in subunit 2 of the ba(3)-type cytochrome oxidase from Thermus thermophilus. The assembly of this site within the periplasmic membrane is believed to be mediated by the copper chaperones Sco and/or PCuAC, but the biological mechanisms are still poorly understood, thereby stimulating interest in the mechanisms of CuA formation from inorganic ions. The formulation of the CuA center as an electron-delocalized Cu(1.5)-Cu(1.5) system implicates both Cu(II) and Cu(I) states in the metalation process. In earlier work we showed that selenomethionine (SeM) substitution of the coordinated M160 residue provided a ligand-directed probe for studying the copper coordination environment via the Se XAS signal, which was particularly useful for interrogating the Cu(I) states where other spectroscopic probes are absent. In the present study we have investigated the formation of mixed-valence CuA and its M160SeM derivative by stopped-flow UV-vis, EPR, and XAS at both Cu and Se edges, while the formation of fully reduced di-Cu(I) CuA has been studied by XAS alone. Our results establish the presence of previously undetected mononuclear intermediates and show important differences from the metalation reactions of purple CuA azurin. XAS spectroscopy at Cu and Se edges has allowed us to extend mechanistic inferences to formation of the di-Cu(I) state which may be more relevant to biological CuA assembly. In particular, we find that T. thermophilus CuA assembles more rapidly than reported for other CuA systems and that the dominant intermediate along the pathway to mixed-valence is a new green species with λ(max) = 460 nm. This intermediate has been isolated in a homogeneous state and shown to be a mononuclear Cu(II)-(His)(Cys)(2) species with no observable Cu(II)-(Met) interaction. Reduction with dithionite generates its Cu(I) homologue which is again mononuclear but now shows a strong interaction with the Met160 thioether. The results are discussed within the framework of the "coupled distortion" model for Cu(II) thiolates and their relevance to biological metalation reactions of the CuA center.

Project description:The R481 residue of cytochrome bo(3) ubiquinol oxidase from E. coli is highly conserved in the heme-copper oxidase superfamily. It has been postulated to serve as part of a proton loading site that regulates proton translocation across the protein matrix of the enzyme. Along these lines, proton pumping efficiency has been demonstrated to be abolished in many R481 mutants. However, R481Q in bo(3) from E. coli has been shown to be fully functional, implying that the positive charge of the arginine is not required for proton translocation [ Puustinen , A. and Wikstrom , M. ( 1999 ) Proc. Natl. Acad. Sci. U.S.A. 96 , 35 - 37 ]. In an effort to delineate the structural role of R481 in the bo(3) oxidase, we used resonance Raman spectroscopy to compare the nonfunctional R481L mutant and the functional R481Q mutant, to the wild type protein. Resonance Raman data of the oxidized and reduced forms of the R481L mutant indicate that the mutation introduces changes to the heme o(3) coordination state, reflecting a change in position and/or coordination of the Cu(B) located on the distal side of heme o(3), although it is approximately 10 A away from R481. In the reduced-CO adduct of R481L, the frequencies of the Fe-CO and C-O stretching modes indicate that, unlike the wild type protein, the Cu(B) is no longer close to the heme-bound CO. In contrast, resonance Raman data obtained from the various oxidation and ligation states of the R481Q mutant are similar to those of the wild type protein, except that the mutation causes an enhancement of the relative intensity of the beta conformer of the CO-adduct, indicating a shift in the equilibrium between the alpha and beta conformers. The current findings, together with crystallographic structural data of heme-copper oxidases, indicate that R481 plays a keystone role in stabilizing the functional structure of the Cu(B) site through a hydrogen bonding network involving ordered water molecules. The implications of these data on the proton translocation mechanism are considered.

Dataset Information

A spectroscopic investigation of a tridentate Cu-complex mimicking the tyrosine-histidine cross-link of cytochrome C oxidase.

Publications

A spectroscopic investigation of a tridentate Cu-complex mimicking the tyrosine-histidine cross-link of cytochrome C oxidase.

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