A Common Mechanism for the Interaction of Nitric Oxide with the Oxidized Binuclear Centre and Oxygen Intermediates of Cytochromec Oxidase

Abstract

The reactions of nitric oxide (NO) with fully oxidized cytochrome c oxidase (O) and the intermediates P and F have been investigated by optical spectroscopy, using both static and kinetic methods. The reaction of NO with O leads to a rapid (ϳ100 s ؊1 ) electron ejection from the binuclear center to cytochrome a and Cu A . The reaction with the intermediates P and F leads to the depletion of these species in slower reactions, yielding the fully oxidized enzyme. The fastest optical change, however, takes place within the dead time of the stopped-flow apparatus (ϳ1 ms), and corresponds to the formation of the F intermediate (580 Cytochrome c oxidase (ferrocytochrome c oxidoreductase, EC 1.9.3.1), the terminal enzyme in the mitochondrial respiratory chain, catalyzes the reduction of molecular oxygen to water (1). This process is coupled to proton translocation across the inner membrane. The enzyme contains four redox-active centers. Electron entry from cytochrome c, the natural substrate, occurs via a diatomic copper center, Cu A . After rapid equilibrium with Fe a , 1 the electron is transferred to Cu B and Fe a3 , which together constitute the binuclear center, where oxygen is reduced.Although oxygen binds with low affinity (10 3 M Ϫ1 ) (2) to reduced Fe a3 , rapid electron transfer from the two reduced metals comprising the binuclear center to molecular oxygen ensures that oxygen remains bound as a peroxy species. In this way, oxygen is kinetically trapped and further reduction can take place (3, 4). The successive steps leading to the formation of water have been studied by different spectroscopic techniques (see Ref. 5 for review). The spectral signatures of two of these intermediates, which exhibit bands at 607 and 580 nm in the difference spectrum with respect to the oxidized enzyme, were first reported by Wikström (6), by reversing the electron transfer reaction, and further characterized by Wikström and Morgan (7). These authors assigned the spectral signatures at 607 and 580 nm to a ferric peroxy (P) and ferryl oxo (F) species, respectively. These assignments, however, have been challenged by a number of authors (8 -10). For example, Resonance Raman results obtained by Proshlyakov et al. (10) suggest that the 607 nm band originates from an oxoferryl structure. On the other hand, the same authors have been unable to identify the putative peroxy species in their system in turnover sustained by H 2 O 2 (11). However, irrespective of the assignments, there seems to exist a general agreement that compound F (580 nm) is a ferryl oxo species and that it is one electron more reduced than compound P (607 nm) (11,12).One of the ways to solve the problem of the identity of the P intermediate could perhaps be through the use of a suitable probe. The possibility that nitric oxide (NO), a powerful reversible inhibitor of cytochrome c oxidase (13-15), can be used as a probe for the binuclear center has been suggested by recent results using enzyme in slow turnover (16 -18). In these experiments, an electron was ejected...

Nitric oxide inhibition of respiration involves both competitive (heme) and noncompetitive (copper) binding to cytochromecoxidase

“…To fit the data in Fig. 2 A, this observation was taken into account, and the model of Antunes et al (43) was expanded to include an additional interaction at a noncompetitive site, consistent with previous observations that NO can react with the oxidized copper of the binuclear center, resulting in oxidation of NO to nitrite (25,26). Fig.…”

“…2). Note that, although a number of iron:oxygen intermediates (e.g., ferryl) are also likely to be present during turnover, the interactions of NO with these oxidized species (26,27) are formally included in the reaction step governed by k 1, and so this added complexity does not affect the conclusions drawn from this model. Additionally, the reaction step governed by k 2 is more complex than indicated because the nitrite dissociation rate depends on the redox state of the binuclear center (29 (43).…”

“…Functional studies initially suggested that NO inhibits by binding reversibly and competitively with oxygen (4-6, 18, 19); however, NO not only reacts rapidly with reduced heme a 3 (20) but also has the capability to react with cupric Cu B (21-24) as well as possibly with the latter in its cuprous state (23). We (25,26) and others (27,28) demonstrated that NO not only binds rapidly to cupric Cu B but also reduces it to the cuprous state, producing nitrite, which is subsequently released from the binuclear center (29). NO similarly interacts rapidly with cupric copper centers in other oxidases (e.g., laccase; refs.…”

On the mechanism and biology of cytochrome oxidase inhibition by nitric oxide

“…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 Cu B ͞heme a 3 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 Cu B , in which NO is initially oxidized, yielding nitrite (14)(15)(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 (6).…”

Cytochrome c oxidase regulates endogenous nitric oxide availability in respiring cells: A possible explanation for hypoxic vasodilation

“…More recent evidence has suggested that cytochrome c oxidase may provide a metabolic route for NO, either by interaction of NO with the reduced enzyme, leading to the formation of N 2 O, (18,19) or by interaction of NO with the oxidized enzyme, forming nitrite (NO 2 Ϫ ; 20,21). Although the NO reductase activity of the enzyme is too slow to constitute a physiological mechanism for the removal of NO (22), there is strong evidence in favor of the oxidation of NO to NO 2 Ϫ both by the purified enzyme (23,24) and by cells (25).…”