Nitric oxide reacts with the single-electron reduced active site of cytochrome c oxidase.
The reduction kinetics of the mutants K354M and D124N of the Paracoccus denitrificans cytochrome oxidase (heme aa(3)) by ruthenium hexamine was investigated by stopped-flow spectrophotometry in the absence/presence of NO. Quick heme a reduction precedes the biphasic heme a(3) reduction, which is extremely slow in the K354M mutant (k(1) = 0.09 +/- 0.01 s(-1); k(2) = 0.005 +/- 0.001 s(-1)) but much faster in the D124N aa(3) (k(1) = 21 +/- 6 s(-1); k(2) = 2.2 +/- 0.5 s(-1)). NO causes a very large increase (>100-fold) in the rate constant of heme a(3) reduction in the K354M mutant but only a approximately 5-fold increase in the D124N mutant. The K354M enzyme reacts rapidly with O(2) when fully reduced but is essentially inactive in turnover; thus, it was proposed that impaired reduction of the active site is the cause of activity loss. Since at saturating [NO], heme a(3) reduction is approximately 100-fold faster than the extremely low turnover rate, we conclude that, contrary to O(2), NO can react not only with the two-electron but also with the single-electron reduced active site. This mechanism would account for the efficient inhibition of cytochrome oxidase activity by NO in the wild-type enzyme, both from P. denitrificans and from beef heart. Results also suggest that the H(+)-conducting K pathway, but not the D pathway, controls the kinetics of the single-electron reduction of the active site.
Giuffrè
Alessandro
A
Department of Biochemical Sciences and CNR Institute of Molecular Biology and Pathology, University of Rome La Sapienza, I-00185 Rome, Italy. alessandro.guiffre@uniroma1.it
Barone
Maria Cecilia
MC
Brunori
Maurizio
M
D'Itri
Emilio
E
Ludwig
Bernd
B
Malatesta
Francesco
F
Müller
Hans-Werner
HW
Sarti
Paolo
P
D001665:Binding Sites; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000378:metabolism; D004583:Electrons; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D009569:Nitric Oxide / Q000737:chemistry* / Q000378:metabolism; D010231:Paracoccus denitrificans / Q000201:enzymology; D011485:Protein Binding; D013997:Time Factors
D009569:Nitric Oxide; D003576:Electron Transport Complex IV