Formation of nitric oxide from nitroxyl anion: role of quinones and ferricytochrome c

Abstract

1. Our previous finding that copper ions oxidize nitroxyl anion released from Angeli's salt to nitric oxide prompted us to examine if copper-containing enzymes shared this property. 2. The copper-containing enzyme, tyrosinase, which catalyses the hydroxylation of monophenols to diphenols and the subsequent oxidation of these to the respective unstable quinone, failed to generate nitric oxide from Angeli's salt by itself, but did so in the presence of tyrosine. 3. L-DOPA, the initial product of the reaction of tyrosinase with tyrosine, was not the active species, since it failed to generate nitric oxide from Angeli's salt. Nevertheless, L-DOPA and two other substrates, namely, catechol and tyramine did produce nitric oxide from Angeli's salt in the presence of tyrosinase, suggesting involvement of the respective unstable quinones. In support, we found that 1,4-benzoquinone produced a powerful nitric oxide signal from Angeli's salt. 4. Coenzyme Q(o), an analogue of ubiquinone, failed to generate nitric oxide from Angeli's salt by itself, but produced a powerful signal in the presence of its mitochondrial complex III cofactor, ferricytochrome c. 5. Experiments conducted on rat aortic rings with the mitochondrial complex III inhibitor, myxothiazol, to determine if this pathway was responsible for the vascular conversion of nitroxyl to nitric oxide were equivocal: relaxation to Angeli's salt was inhibited but so too was that to unrelated relaxants. 6. Thus, certain quinones oxidize nitroxyl to nitric oxide. Further work is required to determine if endogenous quinones contribute to the relaxant actions of nitroxyl donors such as Angeli's salt.

Figure 1

An individual experimental trace showing that tyrosinase (250 u ml⁻¹) alone failed to promote the production of nitric oxide from the nitroxyl generator, Angeli's salt (AS; 0.01 – 10 μM). The additional presence of the substrate tyrosine (100 μM) led, however, to the generation of a powerful nitric oxide signal.

Figure 2

Bar graph showing the ability of tyrosine, L-DOPA, catechol and tyramine (each at 100 μM) either alone (first bar of pair) or in combination with tyrosinase (250 u ml⁻¹; second bar of pair) to promote the production of nitric oxide from the nitroxyl generator, Angeli's salt (10 μM). Each value is the mean±s.e.mean of 5 – 7 observations. ^***P<0.001 indicates a significant generation of nitric oxide.

Figure 3

Bar graph showing the ability of benzoquinone (BZQ), coenzyme Q_o (CQ), duroquinone (DRQ), menadione (MD), hydroquinone (HQ) and pyrogallol (PYR) (all at 100 μM) to promote the production of nitric oxide from the nitroxyl generator, Angeli's salt (10 μM). The ability of superoxide dismutase (SOD; 250 u ml⁻¹) or ascorbate (ASC; 100 μM) to inhibit the generation of nitric oxide by benzoquinone is also shown. Each value is the mean±s.e.mean of 5 – 6 observations. ^***P<0.001 indicates a significant generation of nitric oxide and ^###P<0.001 indicates a significant inhibition by ascorbate.

Figure 4

Bar graph showing the ability of coenzyme Q_o (CQ; 1 mM) either alone or in combination with 100 μM of each of NADH, succinate (Succ) or ferricytochrome c (Cyt) to promote the production of nitric oxide from the nitroxyl generator, Angeli's salt (10 μM). Note that the order of addition is critical for nitric oxide formation, i.e. coenzyme Q_o was effective only when added following pre-reaction of Angeli's salt with ferricytochrome c. The ability of ascorbate (ASC; 100 μM) to inhibit the production of nitric oxide by the combination of coenzyme Q_o and ferricytochrome c is also shown. Each value is the mean±s.e.mean of five observations. ^***P<0.001 indicates a significant generation of nitric oxide and ^###P<0.001 indicates a significant reduction.

Figure 5

An individual experimental trace showing that in the presence of ferricytochrome c (Cyt; 100 μM), coenzyme Q_o (1 mM) promotes the production of nitric oxide from the nitroxyl generator, Angeli's salt (10 μM).

Figure 6

Concentration-response curves showing the effects of pretreating rat aortic rings with the mitochondrial complex III inhibitor, myxothiazol (10 μM; 20 min), on relaxation to (a) Angeli's salt, (b) sodium nitroprusside (SNP), (c) 8-bromo-cGMP, (d) acetylcholine (ACh), (e) atrial natriuretic factor (ANF), (f) papaverine. Each point is the mean±s.e.mean of 6 – 10 observations. ^***P<0.001 indicates a significant difference from control.