Functional properties of the heme propionates in cytochrome c oxidase from Paracoccus denitrificans. Evidence from FTIR difference spectroscopy and site-directed mutagenesis.
By specific (13)C 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 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, 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 cytochromic oxidase. The ring D propionate of heme a(3) might undergo conformational changes or, less likely, act as a proton donor.
Behr
J
J
Max-Planck-Institut für Biophysik, Abteilung Molekulare Membranbiologie, Heinrich-Hoffmann-Strasse 7, D-60528 Frankfurt/M., Germany.
Michel
H
H
Mäntele
W
W
Hellwig
P
P
D019943:Amino Acid Substitution / Q000235:genetics; D001120:Arginine / Q000235:genetics; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006418:Heme / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000235:genetics; D006860:Hydrogen Bonding; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010231:Paracoccus denitrificans / Q000201:enzymology* / Q000235:genetics*; D011422:Propionates / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared; D014364:Tryptophan / Q000235:genetics; D014443:Tyrosine / Q000235:genetics
D011422:Propionates; D011994:Recombinant Proteins; D014443:Tyrosine; D006418:Heme; D006639:Histidine; D014364:Tryptophan; D001120:Arginine; D003576:Electron Transport Complex IV