Suicide inactivation of hydroxylamine oxidoreductase of Nitrosomonas europaea by organohydrazines.
In the presence of a suitable electron acceptor such as mammalian cytochrome c, hydroxylamine oxidoreductase (HAO) from the chemolithotrophic bacterium Nitrosomonas europaea catalyzes the oxidation of hydroxylamine 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 results in reduction of several c-hemes of HAO. The reaction of organohydrazines 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, hydroxylamine or hydrazine. Complete loss of enzyme activity and absorbancy characteristic of native heme P460 of HAO occurred at a 1:1 ratio of phenylhydrazine and HAO. HAO was covalently derivatized by two molecules of [14C]-phenylhydrazine per subunit. Heme P460 was derivatized with high affinity, and an amino acid residue was derivatized with lower affinity. c-Hemes were not derivatized except for the partial reaction of (hydroxyethyl)hydrazine with one heme. As with hydroxylamine and hydrazine, 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 which were generally similar to shifts seen in the reaction of the heme of other hemoproteins with organohydrazines.(ABSTRACT TRUNCATED AT 250 WORDS)
Logan
M S
MS
Department of Genetics and Cell Biology, University of Minnesota, St. Paul 55108, USA.
Hooper
A B
AB
D001665:Binding Sites; D006418:Heme / Q000737:chemistry; D006834:Hydrazines / Q000494:pharmacology*; D007700:Kinetics; D009002:Monomethylhydrazine / Q000494:pharmacology; D009606:Nitrosomonas / Q000201:enzymology*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D010659:Phenylhydrazines / Q000494:pharmacology; D013053:Spectrophotometry
D006834:Hydrazines; D010659:Phenylhydrazines; C030299:phenylhydrazine; D006418:Heme; D010088:Oxidoreductases; C021401:hydroxylamine dehydrogenase; D009002:Monomethylhydrazine