An electron nuclear double resonance investigation of redox-induced electronic structural change at CuA2+ in cytochrome c oxidase.
We measured an electronic change at cysteine ligand(s) of the CuA2+ center brought on by reduction of other metal centers within cytochrome c oxidase, notably cytochrome a. This change specifically manifested itself as a modification in magnetic hyperfine coupling to the beta-protons of the beta-carbons adjacent to the cysteine sulfur in the CuA2+ coordination sphere. The electron nuclear double resonance ENDOR signals of these beta-protons had previously been assigned through study of selectively deuterated yeast oxidase. In the present study the ENDOR signals of the CuA2+ center were compared from the following forms of oxidase: resting (a3+.CuA2+.a3+3.CuB2+); mixed valence, 2-electron-reduced CO-ligated oxidase (a3+.CuA2+.a2+3CO.CuB+), and a more completely reduced mixed-valence CO-ligated oxidase. In agreement with previous studies on 3-electron-reduced oxidase, the latter more completely reduced oxidase showed cytochrome a preferentially reduced with respect to CuA, implying that the majority of paramagnetic CuA2+ centers had reduced cytochrome a partners. The ENDOR-resolved splitting of the beta-proton hyperfine features substantially decreased in going from the first two more oxidized forms to the more fully reduced latter form. Thus, the electronic structure of the CuA2+ center specifically monitored by hyperfine couplings to cysteine protons changed in response to a reductive event elsewhere in the protein. This structural change may correlate with the anticooperative redox interaction recently reported between cytochrome a and CuA.
Fan
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Department of Physics, State University of New York at Albany 12222.
Bank
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Dorr
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Scholes
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D000818:Animals; D001665:Binding Sites; D002245:Carbon Dioxide / Q000378:metabolism; D002417:Cattle; D003300:Copper / Q000378:metabolism*; D003545:Cysteine; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D003576:Electron Transport Complex IV / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D009206:Myocardium / Q000201:enzymology; D011485:Protein Binding; D011487:Protein Conformation
D002245:Carbon Dioxide; D003300:Copper; D003576:Electron Transport Complex IV; D003545:Cysteine