Proton uptake upon anaerobic reduction of the Paracoccus denitrificans cytochrome c oxidase: a kinetic investigation of the K354M and D124N mutants
| Autor: | Forte E, Scandurra FM, Richter OM, D'Itri E, Sarti P, Brunori M, Ludwig B, Giuffrè A. |
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| Rok vydání: | 2004 |
| Zdroj: | Biochemistry (Easton) 43 (2004): 2957–2963. info:cnr-pdr/source/autori:Forte E; Scandurra FM; Richter OM; D'Itri E; Sarti P; Brunori M; Ludwig B; Giuffrè A./titolo:Proton uptake upon anaerobic reduction of the Paracoccus denitrificans cytochrome c oxidase: a kinetic investigation of the K354M and D124N mutants./doi:/rivista:Biochemistry (Easton)/anno:2004/pagina_da:2957/pagina_a:2963/intervallo_pagine:2957–2963/volume:43 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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