Highly Purified Hydroxylamine Oxidoreductase Derived from Nitrosomonas europaea: Some Physicochemical and Enzymatic Properties1

Abstract

Hydroxylamine oxidoreductase [EC 1.7.3.4] of Nitrosomonas europaea was purified to an electrophoretically homogeneous state and some of its properties were studied. The molecular weight of the enzyme as determined by gel filtration on Sephadex G150 and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate is 175,000-180,000, while the minimum molecular weight per heme determined from the dry weight and heme content is 17,500. The enzyme is a C-type cytochrome; its reduced form shows absorption peaks at 418 (gamma peak), 521 (beta peak), 553 (alpha peak), and 460 nm (due to an unidentified chromophore). Although the alpha peak at 553 nm has a shoulder at 559 nm, the enzyme does not posses protoheme or a cytochrome b subunit. It seems likely that the enzyme molecule possess heme c molecules in different states. The enzyme reacts rapidly with various eukaryotic cytochromes c, but does not react with "bacterial-type" cytochromes c. Although the enzyme does not react with cytochrome c-552 (N. europaea), another C-type cytochrome of the organism, cytochrome c-554 (N. europaea) acts as an electron acceptor for the enzyme.

Effect of Salinity on Hydroxylamine Oxidation in a Marine Ammonia-Oxidizing Gammaproteobacterium, Nitrosococcus oceani strain NS58: Molecular and Catalytic Properties of Tetraheme Cytochrome c-554

“…Absorption spectra of the protein in the oxidized state (solid line) and Na2S2O4-reduced state (dotted line) were measured. horse mitochondrial cytochrome c or N. europaea cytochrome c-554 was used, the NS58 HAO showed activity comparable to that of the N. europaea enzyme (12,39). Reactivity with the NS58 cytochrome c-554 was 2.3-fold greater than that with the N. europaea cytochrome c-554.…”

“…Purification of HAO was carried out according to the method of Yamanaka et al (39) with a slight modification. The fraction precipitated by 50-90% (NH4)2SO4 was dissolved in a minimum volume of buffer A, and applied to a column (2×120 cm) of Sephacryl S-200 (Pharmacia) that had been equilibrated with buffer A.…”

Characterization of the gene encoding hydroxylamine oxidoreductase in Nitrosomonas europaea

“…Because of the complexity of HAO, determination of the molecular weight and structure of this enzyme has proven difficult. Depending on the method of determination, estimates of the native molecular weight vary from 150,000 to 200,000 (3,22,28,32). Both two and one subunits have also been proposed (63 and 11 kDa) (12,18).…”

Hydroxylamine Oxidoreductase

“…The differences among these three copies have not been investigated. The isoelectric point was reported as 4.7 3 and 5.3 17 for the oligomeric enzyme, in contrast to the value of 10.7 for cytochrome c 554 . 13 …”

“…Heme content was first determined by the alkaline pyridine hemochrome method. 3,17,18 and was finally confirmed by the peptide digestion study 19 and the nucleotide sequencing. 15 A total metal content of HAO from N. europaea was also determined by plasma emission analysis.…”