Abstract
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.
Highlights
Cytochrome c oxidase (CcOX)1 contains a bimetallic active site where O2 is reduced to H2O
We studied by stopped-flow spectrophotometry the kinetics of reduction of the K354M and the D124N mutants of the P. denitrificans CcOX both in the presence and in the absence of NO
NO is a very efficient, yet reversible, inhibitor of cytochrome c oxidase activity [27, 28], leading to the proposal that it may act as a physiological modulator of cell respiration [29]
Summary
CcOX, cytochrome c oxidase; SVD, singular value decomposition; O, enzyme with oxidized heme a3-CuB site; E, enzyme with a single-electron reduced heme a3-CuB; R, enzyme with a two-electron reduced heme a3-CuB. Mutation of Lys-354 to M within the K pathway yields a virtually inactive enzyme, as shown for the Rhodobacter sphaeroides aa (9 –11), the Escherichia coli bo3 [12], and the Paracoccus denitrificans aa3 [5] This mutation affects primarily, but not exclusively (see Ref. 13), the reductive part of the catalytic cycle [9, 11, 14]; in the absence of O2 and with a large excess of reductant, heme a3 is reduced at an extremely low rate (time scale of several minutes) as compared with the wild type (time scale of tens of milliseconds). We provide evidence for the reaction of NO with E by studying the kinetics of reduction of the K354M and D124N mutants of P. denitrificans CcOX in the presence of NO
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