Mitochondrial Physiology Network 10.9: 33 (2005) - download pdf

Interactions of nitric oxide with mitochondrial cytochrome oxidase : a complete kinetic model requires binding to both haem and copper.

Chris E Cooper, MG Mason, MT Wilson, P Nicholls

Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, Essex, CO4 3SDQ, UK. - ccooper@essex.ac.uk

    The nitric oxide signalling pathway is classically mediated by activation of soluble guanylate cyclase. However, in 1994 it was additionally shown that mitochondrial oxygen consumption by cytochrome oxidase is reversibly inhibited by NO in a manner apparently competitive with the oxygen tension [1]. Inhibition of mitochondrial respiration by NO at cytochrome oxidase has since been implicated in a wide range of physiological processes, [2-3] including redox signalling, oxygen sparing and brain blood flow-metabolism coupling. It was initially suggested that NO inhibited by binding to the ferrous haem a₃ oxygen binding site. However, in 1997 we demonstrated that NO could also interact with an oxidised copper centre in the enzyme (Cu_B) that does not bind oxygen [4]. A recent paper has attempted to explain all the extant literature data within a simple one-site competitive model [5]. Here we report experimental and modelling studies that demonstrate that such a model is inconsistent with NO interactions with the enzyme. Although NO inhibition is always oxygen sensitive, the sensitivity is not consistent with a pure competitive interaction. Non-competitive interactions at a second (Cu_B) site are required and these effects predominate at low oxygen consumption rates and high pO₂. We describe a complete kinetic model of NO inhibition of cytochrome oxidase which, given the cellular NO, O₂ and oxygen consumption rates, describes not only the expected degree of inhibition of the enzyme, but also the nature of the inhibited state. Surprisingly at low NO concentrations (<50 nM) the copper interaction is preferred over the haem even at relatively low oxygen tensions (20 µM). Nitric oxide:copper reactions are therefore crucial in maintaining NO control of mitochondria throughout the in vivo range of mitochondrial oxygen consumption rates and oxygen tensions.

1.  Brown GC, Cooper CE (1994) Nanomolar concentrations of nitric oxide reversibly inhibit synaptosomal cytochrome oxidase respiration by competing with oxygen at cytochrome oxidase. FEBS Lett. 356: 295-298.

2.  Brookes P, Darley-Usmar VM (2002) Hypothesis: the mitochondrial NO(*) signaling pathway, and the transduction of nitrosative to oxidative cell signals: an alternative function for cytochrome c oxidase. Free Radic. Biol. Med. 32: 370-374.

3.  Brown GC (1995) Nitric oxide regulates mitochondrial respiration and cell functions by inhibiting cytochrome oxidase. FEBS Lett. 369: 136-139.

4.  Cooper CE, Torres J, Sharpe MA, Wilson MT (1997) Nitric oxide ejects electrons from the binuclear centre of cytochrome c oxidase by reacting with oxidised copper: a general mechanism for the interaction of copper proteins with nitric oxide? FEBS Lett. 414: 281-284.

5.  Antunes F, Boveris A, Cadenas E (2004) On the mechanism and biology of cytochrome oxidase inhibition by nitric oxide. Proc. Natl. Acad. Sci. USA 101: 16774-16779.