Oxidation of hydroxylamine by cytochrome P-460 of the obligate methylotroph Methylococcus capsulatus Bath.
An enzyme capable of the oxidation of hydroxylamine to nitrite was isolated from the obligate methylotroph Methylococcus capsulatus Bath. The absorption spectra in cell extracts, electron paramagnetic resonance spectra, molecular weight, covalent attachment of heme group to polypeptide, and enzymatic activities suggest that the enzyme is similar to cytochrome P-460, a novel iron-containing protein previously observed only in Nitrosomonas europaea. The native and subunit molecular masses of the M. capsulatus Bath protein were 38,900 and 16,390 Da, respectively; the isoelectric point was 6.98. The enzyme has approximately one iron and one copper atom per subunit. The electron paramagnetic resonance spectrum of the protein showed evidence for a high-spin ferric heme. In contrast to the enzyme from N. europaea, a 13-nm blue shift in the soret band of the ferrocytochrome (463 nm in cell extracts to 450 nm in the final sample) occurred during purification. The amino acid composition and N-terminal amino acid sequence of the enzyme from M. capsulatus Bath was similar but not identical to those of cytochrome P-460 of N. europaea. In cell extracts, the identity of the biological electron acceptor is as yet unestablished. Cytochrome c-555 is able to accept electrons from cytochrome P-460, although the purified enzyme required phenazine methosulfate for maximum hydroxylamine oxidation activity (specific activity, 366 mol of O2 per s per mol of enzyme). Hydroxylamine oxidation rates were stimulated approximately 2-fold by 1 mM cyanide and 1.5-fold by 0.1 mM 8-hydroxyquinoline.
Zahn
J A
JA
Department of Microbiology, Immunology, and Preventive Medicine, Iowa State University, Ames 50011.
Duncan
C
C
DiSpirito
A A
AA
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000641:Ammonia / Q000378:metabolism; D003580:Cytochromes / Q000302:isolation & purification / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D006418:Heme / Q000032:analysis; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000378:metabolism*; D007526:Isoelectric Point; D008670:Metals / Q000032:analysis; D008697:Methane / Q000378:metabolism; D008772:Methylococcaceae / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009573:Nitrites / Q000378:metabolism*; D013053:Spectrophotometry
D000596:Amino Acids; D003580:Cytochromes; D006898:Hydroxylamines; D008670:Metals; D009573:Nitrites; D019811:Hydroxylamine; D006418:Heme; C080650:cytochrome P-460; D000641:Ammonia; D008697:Methane