Mitochondrial Physiology Network 10.9: 34 (2005) - download pdf

Differential sensitivity of cytochrome c oxidase and guanylate cyclase to endogenous NO at physiological oxygen concentrations.

F Rodríguez-Juárez, E Aguirre, Susana Cadenas

Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), Melchor Fernández Almagro 3, 28029 Madrid, Spain. - scadenas@cnic.es

Nitric oxide (NO) stimulates cGMP production by binding to the haem iron of soluble guanylate cyclase. NO also binds to the binuclear center of cytochrome c oxidase reversibly inhibiting mitochondrial oxygen consumption in competition with oxygen [1,2]. Recent work suggests that guanylate cyclase is significantly more sensitive to being activated by NO than cytochrome c oxidase is to being inhibited at physiological p_O2 [3]. The aim of this study was to determine the sensitivity of both enzymes to endogenously produced NO at physiological oxygen concentrations (30 µM O₂). We used HEK 293 cells transfected with the inducible isoform of the NO synthase gene under the control of a tetracycline-inducible promoter. In this system, NO is generated inside the cells at different levels for extended periods of time. The amount of NO produced was within the physiopathological range (up to 1.3 µM). NO-stimulated cGMP production was measured at 30 µM O₂ in hypoxic chambers, while mitochondrial oxygen consumption was deter-mined by high-resolution respirometry (OROBOROS Oxygraph-2k). Under physiological conditions of p_O2, the NO concentration giving half-maximal activation (EC₅₀) of guanylate cyclase was around 3 nM, whereas that required to achieve 50 % inhibition of respiration (IC₅₀) was determined to be 160 nM from a value of 14 pmol∙s^-1∙10^-6 cells for controls. These data show that, in our system, the IC₅₀ of cytochrome c oxidase is more than 50-fold higher than the EC₅₀ of soluble guanylate cyclase to endogenous NO, thus confirming previous studies using NO donors which suggest that the latter enzyme is more sensitive than mitochondrial respiration to NO at physiological oxygen pressure.

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. Cleeter MWJ, Cooper JM, Darley-Usmar VM, Moncada S, Shapira AHV (1994) Reversible inhibition of cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, by nitric oxide. Implications for neurodegenerative diseases. FEBS Lett. 345: 50-54.

3.  Bellamy TC, Griffiths C, Garthwaite J (2002) Differential sensitivity of guanylyl cyclase and mitochondrial respiration to nitric oxide measured using clamped concentrations. J. Biol. Chem. 277: 31801-31807.