Spectroscopic evidence for a photosensitive oxygenated state of ammonia mono-oxygenase

Abstract

Photoinactivation of NH3 oxidation by Nitrosomonas europaea cells by near-UV light was confirmed and further shown to occur with the same rate constant as loss of bromoethane-oxidation activity. Hydroxylamine oxidation was much less photosensitive. Protection against inactivation was afforded by anaerobiosis, organic substrates of ammonia monooxygenase such as bromoethane, or metal-ion-chelating agents such as thiourea. The presence of 10 mM NH4+ or 1 mM hydroxylamine made little difference, whereas hydrazine had a potentiating effect. Illumination of cells also caused a bleaching in the absorption spectrum around 380 nm, along with changes in the cytochrome .gamma.-band region. Similar effects below 400 nm were obtained when organic substrates and inhibitors of the monooxygenase were added to cells in the dark. The Cu proteins hemocyanin and tyrosinase have a photosensitive oxygenated state with a near-UV absorption band of similar half-width. They also have a sensitivity to chelating agents similar to that of ammonia monoxygenase. The experimental results are explained in terms of a 3-stage catalytic cycle analogous to that for tyrosinase. In resting cells most of the enzyme is believed to be in an oxygenated (Oxy) form, which absorbs maximally at 378 nm and is photosensitive. In the presence of a substrate. One O atom is inserted into the substrate and the other is reduced to water, leaving the enzyme in an oxidized (Met) state. This is followed by a 2-electron reduction of the proposed binuclear Cu site to give a reduced (Deoxy) state, which can bind O2 to complete the cycle.