Involvement of a novel hydroxylamine oxidoreductase in anaerobic ammonium oxidation.
In this study a novel hydroxylamine oxidoreductase (HAO) was purified and characterized from an anaerobic ammonium-oxidizing (Anammox) enrichment culture. The enzyme, which constituted about 9% of the protein mass in the soluble fraction of the cell extract, was able to oxidize hydroxylamine and hydrazine. When phenazine methosulfate and methylthiazolyltetrazolium bromide were used as electron acceptors, a V(max) [21 and 1.1 micromol min(-)(1) (mg of protein)(-)(1)] and K(m) (26 and 18 microM) for hydroxylamine and hydrazine were determined, respectively. The hydroxylamine oxidoreductase is a trimer and contains about 26 hemes per 183 kDa. As deduced from UV/vis spectra, hydroxylamine reduced more and different cytochromes than hydrazine. The dithionite-reduced spectrum showed an unusual 468 nm peak. Inhibition experiments with H(2)O(2) showed that hydroxylamine bound to this P-468 cytochrome, which is assumed to be the putative substrate binding site. Cyanide and hydrazine inhibited the oxidation of hydroxylamine. The amino acid sequences of several peptide fragments of HAO from Anammox showed a clear difference with the deduced amino acid sequence of HAO from the aerobic ammonia-oxidizing bacterium Nitrosomonas europaea. In EPR spectra of the Anammox HAO, two g-values (g(z)() = 2.37 and 2.42) were observed, which were not present in HAO of N. europaea.
Schalk
J
J
Department of Biotechnology, Kluyver Laboratory, Delft University of Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.
de Vries
S
S
Kuenen
J G
JG
Jetten
M S
MS
D001426:Bacterial Proteins / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006834:Hydrazines / Q000378:metabolism; D019811:Hydroxylamine / Q000378:metabolism; D007700:Kinetics; D009606:Nitrosomonas / Q000201:enzymology; D010088:Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification; D011487:Protein Conformation; D000644:Quaternary Ammonium Compounds / Q000378:metabolism*; D020539:Sequence Analysis, Protein; D013053:Spectrophotometry; D013379:Substrate Specificity
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D006834:Hydrazines; D000644:Quaternary Ammonium Compounds; C029424:hydrazine; D019811:Hydroxylamine; D010088:Oxidoreductases; C021401:hydroxylamine dehydrogenase