Hydroxylamine oxidoreductase from Nitrosomonas: inactivation by hydrogen peroxide

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Abstract
Incubation of hydroxylamine oxidoreductase of N. [europaea] with H2O2 resulted in the rapid and irreversible loss of the ability to catalyze the dehydrogenation of hydroxylamine in the presence of electron acceptors such as phenazine methosulfate. The rate of the reaction was dependent on the concentration of enzyme and H2O2. Inactivation occurred most rapidly at pH values between 9-10. Inactivation of the enzyme by H2O2 did not result in alteration of the absorption spectrum of the oxidized form of the enzyme or dithionite-reduced enzyme cytochromes with .alpha. maxima in the wavelength range 540-570 nm, indicating that those cytochromes were not directly involved in the dehydrogenase step. Unlike the active enzyme, cytochromes with .alpha. maxima in the wavelength range 540-570 nm were not reducible by hydroxylamine in the inactivated enzyme. The dithionite-induced absorption maximum at 460 nm (cytochrome P 460), present in the active enzyme, was lost upon inactivation of the enzyme. This is the 1st direct indication of the involvement of cytochrome P 460 in the action of hydroxylamine oxidoreductase. Protection from inactivation was afforded by substrates for the reduction of enzyme cytochrome, hydrazine and N-methylhydroxylamine; metal binding agents, KCN, 1,2-dihydroxybenzene-3,5-disulfonate and hydroxyurea; reductants, o-dianisidine, p-phenylenediamine, hydroquinone, pyrogallol and dithiothreitol; electron acceptors, phenazine methosulfate and 2,6-dichlorophenolindophenol; and the singlet oxygen trapping agent, 1,3-diphenylfuran. Scavengers of superoxide anion or hydroxyl radical did not protect the enzyme from inactivation.