Free radical production from the aerobic oxidation of reduced pyridine nucleotides catalysed by phenazine derivatives

Abstract

Free radical production from the reaction of reduced pyridine nucleotides with phenazine derivatives in aerobic media at pH 7.5 has been studied by ESR spectroscopy and the ESR technique of spin trapping. With the spin trapping agent, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), the oxidation of NADH and NADPH catalysed by phenazine methosulphate, phenazine ethosulphate and 1-methoxyphenazine methosulphate gave exclusively the hydroxyl radical spin adduct of DMPO, 2-hydroxy-5,5-dimethylpyrrolidino-1-oxyl (DMPO-OH). DMPO-OH production was inhibited from these systems by catalase and sodium benzoate whereas superoxide dismutase gave a small increase in the rate of DMPO-OH production. NADH gives a higher rate of DMPO-OH production than NADPH with initial rates of DMPO-OH production in the order 1-methoxyphenazine methosulphate > phenazine ethosulphate > phenazine methosulphate. However, for an oxygen-limited system, the maximum DMPO-OH concentration attained varied in the order 1-methoxyphenazine methosulphate > phenazine methosulphate > phenazine ethosulphate. DMPO-OH production occurred in both the aerobic and anaerobic phases of the reaction with these phenazine derivatives. A similar system with pyocyanine gave DMPO-OH and the superoxide spin adduct of DMPO, 2-hydroperoxy-5,5-dimethylpyrrolidino-l-oxyl (DMPO-OOH). Addition of superoxide dismutase to this system stimulated the rate of DMPO-OH production and inhibited DMPO-OOH production. Addition of catalase and sodium benzoate decreased the production of DMPO-OH only. No DMPO-OH production was observed in the anaerobic phase of the reaction. The auto-oxidation of fully reduced phenazine methosulphate, 5,10-methylhydrophenazine methosulphate, produced the phenazine methosulphate radical cation PMSH +· and DMPO-OH in the presence of DMPO. A mechanism for the auto-oxidation of reduced phenazine derivatives is proposed where superoxide production occurs in discrete steps in the auto-oxidation of fully reduced pyocyanine whereas for the auto-oxidation of fully reduced phenazine methosulphate, phenazine ethosulphate and 1-methyoxyphenazine methosulphate, the production of superoxide appears masked by the rapid further reduction to hydrogen peroxide.