（PNAS ）November 30, 2004，vol. 101 ： 16774-16779
On the mechanism and biology of cytochrome oxidase inhibition by nitric oxide
The detailed molecular mechanism for the reversible inhibition of mitochondrial respiration by NO has puzzled investigators: The rate constants for the binding of NO and O2 to the reduced binuclear center CuB/a3 of cytochrome oxidase (COX) are similar, and NO is able to dissociate slowly from this center whereas O2 is kinetically trapped, which altogether seems to favor the complex of COX with O2 over the complex of COX with NO. Paradoxically, the inhibition of COX by NO is observed at high ratios of O2 to NO (in the 40–500 range) and is very fast (seconds or faster). In this work, we used simple mathematical models to investigate this paradox and other important biological questions concerning the inhibition of COX by NO. The results showed that all known features of the inhibition of COX by NO can be accounted for by a direct competition between NO and O2 for the reduced binuclear center CuB/a3 of COX. Besides conciliating apparently contradictory data, this work provided an explanation for the so-called excess capacity of COX by showing that the COX activity found in tissues actually is optimized to avoid an excessive inhibition of mitochondrial respiration by NO, allowing a moderate, but not excessive, overlap between the roles of NO in COX inhibition and in cellular signaling. In pathological situations such as COX-deficiency diseases and chronic inflammation, an excessive inhibition of the mitochondrial respiration is predicted.
The paradigm that the respiratory chain is regulated by ADP and O2 was recently updated to include the reversible inhibition of cytochrome oxidase (COX) by nitric oxide (NO) (1–6). The physiological role and the detailed molecular mechanism of this inhibition, as well as the reason for the apparent excess content of COX compared with other mitochondrial complexes, are fundamental questions of mitochondrial biochemistry that remain unsolved. Concerning the mechanism, the problem is particularly puzzling because the known characteristics of COX inhibition by NO are difficult to conciliate with the available kinetic rate constants for this inhibition. On the one hand, COX is inhibited rapidly, within a time scale of seconds, with half-inhibition of respiration attained at O2/NO ratios in the 40–500 range (4, 7), which apparently indicates that the interaction of COX with NO is stronger than that of COX with O2 and very fast. On the other hand, the rate constants for the binding of O2 and NO with the fully reduced binuclear center (CuB/a3) of COX are similar [1.4 x 108 and 4 x 107 to 1 x 108 M–1·s–1 (9, 10), respectively] and NO dissociates slowly from this center (0.01–0.13 s–1) (11, 12), whereas the apparent dissociation rate constant of O2 with COX is virtually zero because, during the initial steps of reduction of oxygen by COX, oxygen is kinetically trapped . Accordingly, several investigators have noticed that the potent reversible inhibition of COX by NO cannot be based on a simple competition between NO and O2 (6, 7, 11, 13), and alternative mechanisms have been proposed: Besides binding to the two-electron-reduced CuB/heme a3 center, NO would bind to a single-electron-reduced CuB/heme a3, either reduced CuB (13) or reduced heme a3 (11), for which O2 has a low affinity . Originally, these proposals were supported by mathematical models, but the experimental support is missing. Recently, the use of a bacterial mutant in which the reduction of COX is very slow allowed researchers to observe the binding of NO to the single-electron-reduced CuB/heme a3 center (14), but whether this binding is effective in the wild-type protein remains unknown. Also recently, a mechanism for the inhibition of COX was proposed in which NO reacts with the oxidized form of CuB, in which NO is initially oxidized, yielding nitrite (14–16). However, this inhibition is not competitive with oxygen and is not reversible by light as observed in cells, so it is generally accepted that the inhibition of COX by low physiological levels of NO involves binding, rather than reaction of NO with COX .
作者:admin@医学,生命科学 2011-08-28 08:09