Review
Nitric oxide and cytochrome oxidase: substrate, inhibitor or effector?

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Abstract

Endogenously produced nitric oxide (NO) controls oxygen consumption by inhibiting cytochrome c oxidase, the terminal electron acceptor of the mitochondrial electron transport chain. The oxygen-binding site of the enzyme is an iron/copper (haem a3/CuB) binuclear centre. At high substrate (ferrocytochrome c) concentrations, NO binds reversibly to the reduced iron in competition with oxygen. At low substrate concentrations, NO binds to the oxidized copper. Inhibition at the haem iron site is relieved by dissociation of the NO from the reduced iron. Inhibition at the copper site is relieved by oxidation of the bound NO and subsequent dissociation of nitrite from the enzyme. Therefore, NO can be a substrate, inhibitor or effector of cytochrome oxidase, depending on cellular conditions.

Section snippets

Does NO inhibit cytochrome oxidase in vitro?

The 1994 studies 10., 11., 12. demonstrated that nanomolar concentrations of exogenously added NO reversibly inhibited mitochondrial cytochrome in a competitive manner with oxygen. The effect occurred in the purified beef heart enzyme, isolated skeletal muscle mitochondria, rat liver mitochondria and isolated brain nerve terminals. It quickly became obvious that this inhibition was neither species nor tissue specific, and similar effects were observed in many different cell types (reviewed in

Does NO inhibit cytochrome oxidase in vivo?

The body's own defence mechanisms provide indirect evidence for the potential of NO to inhibit cytochrome oxidase in vivo. Knockout mice lacking myoglobin have an increased susceptibility to NO inhibition of cardiac function [25], suggesting that the NO oxidase function of oxymyoglobin protects cardiomyocytes from NO inhibition of the oxidase [26]. The induction of bacterial intracellular haemoglobins in response to NO indicates that a similar defence mechanism exists for invading pathogens in

NO binding to the reduced enzyme: NO + Fe2+ ⇔ Fe2+NO

NO binds tightly to the reduced iron of haem proteins [2]. There is no crystal structure of the NO complex; comparisons with the closely related CO complex (Fig. 3) suggest that NO probably binds to the iron in the distal pocket with the N closest to the iron and the O close to, but not directly bonding, the

CuB. NO binding to reduced cytochrome oxidase has been demonstrated by optical [3] and EPR [6] spectroscopy. The on rate [33] is fast (108 m−1 s−1 at 20°C), whereas the off rate [34] is slow

NO binding to the oxidized enzyme: NO + Cu2+ ⇔ Cu+NO+

NO, unlike CO or O2, has the ability to bind oxidized (ferric) as well as reduced (ferrous) haem iron. In addition, it has long been known that NO has the ability to bind oxidized cytochrome oxidase. However, the interaction between NO and this enzyme is via the oxidized copper centre (CuB) in the binuclear centre, not the iron 4., 5.. The spectroscopic evidence suggested that the initial interaction of NO with Cu2+ led to the subsequent formation of a Cu+NO+ complex [37]. In effect, the NO

How many NO molecules can simultaneously interact with cytochrome oxidase?

From the discussion so far one might suppose that two molecules of NO could combine simultaneously with the cytochrome oxidase binuclear centre (one at the copper and one at the iron). In fact, it is not clear whether this occurs under physiological conditions.

At high NO concentrations, there is some EPR [5], infrared [44] and flash photolysis [45] spectroscopic evidence for two molecules of NO bound to the fully reduced (ferrous and/or cuprous) binuclear centre. However, the second site

Is NO a substrate for cytochrome oxidase?

In addition to passive, reversible binding, NO has the potential to be metabolized by cytochrome oxidase. Low concentrations of NO at physiological pO2 are highly stable – the pathways for removal of NO (to reverse its signalling role) are unclear and it has been suggested that mitochondria (and cytochrome oxidase in particular) could play a role [24]. Two reactions have been suggested to occur – the reduction of NO to N2O by the reduced enzyme [48], and the oxidation of NO to nitrite by the

Is NO a product of cytochrome oxidase?

Nitrite reductase activity generates NO in bacterial haem and copper enzymes [51]. For mammalian cytochrome oxidase, high concentrations of nitrite in the presence of reductants can generate the ferrous haem a3 nitrosyl complex [5]. This Fe2+NO complex has an off rate of 0.01 s−1 [34]; therefore, in principle, cytochrome oxidase can act as a slow nitrite reductase, producing NO at this rate. However, the affinity of nitrite for the enzyme is in the millimolar range 52., 53., a value unlikely

Interactions of other nitrogen oxides with cytochrome oxidase

As well as NO, a variety of other reactive nitrogen species (RNS) have been shown to interact with cytochrome oxidase. These include nitroxyl anion (NO) 54., 55. and peroxynitrite (ONOO) [56]. Peroxynitrite is detoxified by cytochrome oxidase to NO (reduced enzyme) and nitrite (oxidized enzyme) and, at high levels, it can cause irreversible damage to the enzyme. This probably occurs via a combination of tyrosine nitration [57] and damage to the prosthetic groups [56]. One consequence is to

Conclusion

What is the dominant mechanism of NO interaction with mammalian mitochondrial oxidase in vivo? The variety of reactions described above might lead to possible confusion as to what exactly are the physiologically relevant reactions of NO with cytochrome oxidase. The only reactions of NO fast enough to be relevant to the enzyme turnover are those between NO and reduced haem a3 (reaction 1 in Table 1), and between NO and oxidized CuB (reaction 2 in Table 1). In an elegant set of recent experiments

Acknowledgements

I thank Mike Wilson and Peter Nicholls (University of Essex, UK), and Martyn Sharpe (Institute of Neurology, London), for helpful discussions.

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