Proton uptake upon anaerobic reduction of the Paracoccus denitrificans cytochrome c oxidase: a kinetic investigation of the K354M and D124N mutants.
The kinetics and stoichiometry of the redox-linked protonation of the soluble Paracoccus denitrificans cytochrome c oxidase were investigated at pH = 7.2-7.5 by multiwavelength stopped-flow spectroscopy, using the pH indicator phenol red. We compared the wild-type enzyme with the K354M and the D124N subunit I mutants, in which the K- and D-proton-conducting pathways are impaired, respectively. Upon anaerobic reduction by Ru-II hexamine, the wild-type enzyme binds 3.3 +/- 0.6 H(+)/aa(3), i.e., approximately 1 H(+) in excess over beef heart oxidase under similar conditions and the D124N mutant 3.2 +/- 0.5 H(+)/aa(3). In contrast, in the K354M mutant, in which the reduction of heme a(3)-Cu(B) is severely impaired, approximately 0.8 H(+) is promptly bound synchronously with the reduction of heme a, followed by a much slower protonation associated with the retarded reduction of the heme a(3)-Cu(B) site. These results indicate that complete reduction of heme a (and Cu(A)) is coupled to the uptake of approximately 0.8 H(+), which is independent of both H(+)-pathways, whereas the subsequent reduction of the heme a(3)-Cu(B) site is associated with the uptake of approximately 2.5 H(+) transferred (at least partially) through the K-pathway. On the basis of these results, the possible involvement of the D-pathway in the redox-linked protonation of cytochrome c oxidase is discussed.
Department of Biochemical Sciences and CNR Institute of Molecular Biology and Pathology, University of Rome La Sapienza, I-00185 Rome, Italy.
Elena
E
Forte
Francesca Maria
FM
Scandurra
Oliver-Matthias H
OM
Richter
Emilio
E
D'Itri
Paolo
P
Sarti
Maurizio
M
Brunori
Bernd
B
Ludwig
Alessandro
A
Giuffrè
D019943:Amino Acid Substitution / Q000235:genetics*; D000693:Anaerobiosis; D001216:Asparagine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D002138:Calibration; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000235:genetics* / Q000592:standards; D006418:Heme / Q000031:analogs & derivatives* / Q000737:chemistry; D008239:Lysine / Q000235:genetics; D008715:Methionine / Q000235:genetics; D010084:Oxidation-Reduction; D010231:Paracoccus denitrificans / Q000201:enzymology* / Q000235:genetics*; D010637:Phenolsulfonphthalein / Q000592:standards; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics; D011522:Protons; D013053:Spectrophotometry / Q000592:standards
D021122:Protein Subunits; D011522:Protons; C027728:heme a; D001224:Aspartic Acid; D006418:Heme; D001216:Asparagine; D008715:Methionine; D003576:Electron Transport Complex IV; D010637:Phenolsulfonphthalein; D008239:Lysine