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+/aa3, i.e., approximately 1 H+ in excess over beef heart oxidase under similar conditions and the D124N mutant 3.2 +/- 0.5 H+/aa3. In contrast, in the K354M mutant, in which the reduction of heme a3-CuB is severely impaired, 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 a3-CuB site. These results indicate that complete reduction of heme a (and CuA) is coupled to the uptake of 0.8 H+, which is independent of both H+-pathways, whereas the subsequent reduction of the heme a3-CuB site is associated with the uptake of 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.
Proton uptake upon anaerobic reduction of the Paracoccus denitrificans cytochrome c oxidase: a kinetic investigation of the K354M and D124N mutants.
2004
Abstract
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+/aa3, i.e., approximately 1 H+ in excess over beef heart oxidase under similar conditions and the D124N mutant 3.2 +/- 0.5 H+/aa3. In contrast, in the K354M mutant, in which the reduction of heme a3-CuB is severely impaired, 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 a3-CuB site. These results indicate that complete reduction of heme a (and CuA) is coupled to the uptake of 0.8 H+, which is independent of both H+-pathways, whereas the subsequent reduction of the heme a3-CuB site is associated with the uptake of 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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
