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 aa3) by ruthenium hexamine was investigated by stopped-flow spectrophotometry in the absence/presence of NO. Quick heme a reduction precedes the biphasic heme a3 reduction, which is extremely slow in the K354M mutant (k1 = 0.09 ± 0.01 s-1; k2 = 0.005 ± 0.001 s-1) but much faster in the D124N aa3 (k1 = 21 ± 6 s-1; k2 = 2.2 ± 0.5 s-1). NO causes a very large increase (> 100-fold) in the rate constant of heme a3 reduction in the K354M mutant but only a ~5-fold increase in the D124N mutant. The K354M enzyme reacts rapidly with O2 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 a3 reduction is ~100-fold faster than the extremely low turnover rate, we conclude that, contrary to O2, 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.