Catalysis of nitrofuran redox-cycling and superoxide anion production by heart lipoamide dehydrogenase.

Heart lipoamide dehydrogenase (LADH) catalyzed redox-cycling and O2-. production by (5-nitro-2-furfurylidene)amino derivatives using NADH as electron donor. NADH was a much more effective electron donor than NADPH for the nitroreductase activity. O2-. production was demonstrated by cytochrome c reduction, adrenochrome formation and the effect of superoxide dismutase. Under optimum conditions, nitroreductase activity was about 1% of LADH activity. One electron oxygen reduction and NADH oxidation correlated in 2:1 stoichiometry. The nitroreductase kinetics was in accordance with an ordered bi-bi mechanism. Nitrofuran derivatives bearing unsaturated five- or six-membered nitrogen heterocycles were more effective substrates than those bearing other groups, namely nifurtimox, nitrofurazone, nitrofurantoin and 5-nitro-2-furoic acid. Other nitro compounds (chloramphenicol, benznidazole, 2-nitroimidazole and 5-nitroindole) were ineffective. With the triazole, traizine and imidazole nitrofuran derivatives, the nitroreductase pH curve showed a maximum at pH 8.8, different from the pH optimum for the lipoamide reductase and diaphorase activities. Spectroscopic observations demonstrated pH-dependent structural changes in the triazole(I) and triazine derivatives which would affect their behavior as nitroreductase substrates. The nitroreductase activity was inhibited by p-chloromercuribenzoate and enhanced by cadmium and arsenite, whereas the NADH-induced LADH inactivation failed to affect the nitroreductase activity. In the absence of oxygen. LADH catalyzed nitrofuran reduction to products more reduced than the nitroanion, which were not reoxidized by oxygen. The anaerobic nitrofuran reduction was inhibited by cadmium and arsenite. The assayed nitrofuran compounds did not inhibit LADH lipoamide reductase activity, at variance with their action on glutathione reductase (Grinblat et al., Biochem Pharmacol 38: 767-772, 1989).