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 iso lated from the obligate methylotroph Methylococcus capsulatus Bath. Th e absorption spectra in cell extracts, electron paramagnetic resonance spectra, molecular weight, covalent attachment of heme group to polyp eptide, 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 masse s of the M. capsulatus Bath protein were 38,900 and 16,390 Da, respect ively; the isoelectric point was 6.98. The enzyme has approximately on e iron and one copper atom per subunit. The electron paramagnetic reso nance spectrum of the protein showed evidence for a high-spin ferric h eme. 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. c apsulatus Bath was similar but not identical to those of cytochrome P- 460 of N. europaea. In cell extracts, the identity of the biological e lectron acceptor is as yet unestablished. Cytochrome c-555 is able to accept electrons from cytochrome P-460, although the purified enzyme r equired phenazine methosulfate for maximum hydroxylamine oxidation act ivity (specific activity, 366 mol of O-2 per s per mol of enzyme). Hyd roxylamine oxidation rates were stimulated approximately 2-fold by 1 m M cyanide and 1.5-fold by 0.1 mM 8-hydroxyquinoline.