Functional Properties of the Heme Propionates in Cytochrome c Oxidase from Paracoccus denitrificans. Evidence from FTIR Difference Spectroscopy and Site-Directed Mutagenesis

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• 作者：Julia Behr ; Hartmut Michel ; Werner M&auml ; ntele ; and Petra Hellwig
• 刊名：Biochemistry
• 出版年：2000
• 出版时间：February 15, 2000
• 年：2000
• 卷：39
• 期：6
• 页码：1356 - 1363
• 全文大小：141K
• 年卷期：v.39,no.6(February 15, 2000)
• ISSN：1520-4995

文摘

By specific ¹³C labeling of the heme propionates, four bands in the reduced-minus-oxidizedFTIR difference spectrum of cytochrome c oxidase from Paracoccus denitrificans have been assigned tothe heme propionates [Behr, J., Hellwig, P., Mäntele, W., and Michel, H. (1998) Biochemistry 37, 7400-7406]. To attribute these signals to the individual propionates, we have constructed seven cytochrome coxidase variants using site-directed mutagenesis of subunit I. The mutant enzymes W87Y, W87F, W164F,H403A, Y406F, R473K, and R474K were characterized by measurement of enzymatic turnover, protonpumping activity, and Vis and FTIR spectroscopy. Whereas the mutant enzymes W164F and Y406Fwere found to be structurally altered, the other cytochrome c oxidase variants were suitable for bandassignment in the infrared. Reduced-minus-oxidized FTIR difference spectra of the mutant enzymes wereused to identify the ring D propionate of heme a as a likely proton acceptor upon reduction of cytochromec oxidase. The ring D propionate of heme a₃ might undergo conformational changes or, less likely, act asa proton donor.