SUICIDE INACTIVATION OF HYDROXYLAMINE OXIDOREDUCTASE OF NITROSOMONAS-EUROPAEA BY ORGANOHYDRAZINES

In the presence of a suitable electron acceptor such as mammalian cyto chrome c, hydroxylamine oxidoreductase (HAO) from the chemolithotrophi c bacterium Nitrosomonas europaea catalyzes the oxidation of hydroxyla mine or hydrazine to nitrite or dinitrogen, respectively. Each subunit of HAO contains 7 c-hemes and a chromophore of the active site called heme P460, a c-heme bridged from a methylene carbon to a ring carbon of a tyrosine of the peptide chain. Reaction with either substrate res ults in reduction of several c-hemes of HAO. The reaction of organohyd razines with HAO was investigated in this work. HAO was inactivated by (phenyl-, (methyl-, or (hydroxyethyl)hydrazine. The process followed first order kinetics and was inhibited by the substrates, hydroxylamin e or hydrazine. Complete loss of enzyme activity and absorbancy charac teristic of native heme P460 of HAO occurred at a 1:1 ratio of phenylh ydrazine and HAO. HAO was covalently derivatized by two molecules of [ C-14]phenylhydrazine per subunit. Heme P460 was derivatized with high affinity, and an amino acid residue was derivatized with lower affinit y. c-Hemes were not derivatized except for the partial reaction of (hy droxyethyl)hydrazine with one heme. As with hydroxylamine and hydrazin e, incubation with organohydrazines resulted in reduction of c-heme of HAO. Derivatized minus native optical difference spectra of ferric or ferrous HAO revealed changes in the optical properties of heme P460 w hich were generally similar to shifts seen in the reaction of the heme of other hemoproteins with organohydrazines. The data indicate that o rganohydrazines are suicide substrates of HAO and constitute direct ev idence that P460 is at the active site. The data also establish condit ions for the use of organohydrazines as probes for structural and mech anistic analysis of HAO.