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

By specific 13C labeling of the heme propionates, four bands in the reduced-minus-oxidized FTIR difference spectrum of cytochrome c oxidase from Paracoccus denitrificans have been assigned to the heme propionates [Behr, J., Hellwig, P., Mäntele, W., and Michel, H. (1998) Biochemistry 37, 7400−7406]....

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Published in:Biochemistry (Easton) 2000-02, Vol.39 (6), p.1356-1363
Main Authors: Behr, Julia, Michel, Hartmut, Mäntele, Werner, Hellwig, Petra
Format: Article
Language:English
Subjects:
Amino Acid Substitution - genetics
Arginine - genetics
Electron Transport Complex IV - chemistry
Electron Transport Complex IV - genetics
Electron Transport Complex IV - metabolism
Heme - analogs & derivatives
Heme - chemistry
Heme - genetics
Heme - metabolism
Histidine - genetics
Hydrogen Bonding
Mutagenesis, Site-Directed
Oxidation-Reduction
Paracoccus denitrificans
Paracoccus denitrificans - enzymology
Paracoccus denitrificans - genetics
Propionates - chemistry
Propionates - metabolism
Recombinant Proteins - chemistry
Spectroscopy, Fourier Transform Infrared
Tryptophan - genetics
Tyrosine - genetics
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Summary:By specific 13C labeling of the heme propionates, four bands in the reduced-minus-oxidized FTIR difference spectrum of cytochrome c oxidase from Paracoccus denitrificans have been assigned to the 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 c oxidase 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, proton pumping activity, and Vis and FTIR spectroscopy. Whereas the mutant enzymes W164F and Y406F were found to be structurally altered, the other cytochrome c oxidase variants were suitable for band assignment in the infrared. Reduced-minus-oxidized FTIR difference spectra of the mutant enzymes were used to identify the ring D propionate of heme a as a likely proton acceptor upon reduction of cytochrome c oxidase. The ring D propionate of heme a 3 might undergo conformational changes or, less likely, act as a proton donor.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi991504g