Cytochrome P-460 of Nitrosomonas europaea: Further Purification and Further Characterization

Abstract

Cytochrome P-460 of Nitrosomonas europaea [Erickson, R.H. and Hooper, A.B. (1972) Biochim. Biophys. Acta 275, 231-244] was further purified to an electrophoretically homogeneous state. The cytochrome molecule was composed of three molecules of subunits with Mr of 17,300-18,500, and contained three atoms of iron, which seemed to be heme iron, and six cysteine residues, but did not contain nonheme iron or inorganic sulfide. The cytochrome showed absorption peaks at 460 and 688 nm with a broad shoulder at 635 nm in the reduced form. The ESR spectrum of ferricytochrome P-460 showed signals at g = 5.91, 5.63, and 1.99, indicating that the protein was a high spin hemoprotein. The heme of the cytochrome was not cleaved by the methods which were available for cleavage of heme c. The pyridine ferrohemochrome of the hemoprotein did not show the distinct alpha and beta peaks which are shown by the ferrohemochromes of many other cytochromes so far known. The N-terminal amino acid sequence of cytochrome P-460 differed from that of hydroxylamine oxidoreductase. Therefore, cytochrome P-460 did not seem to be the solubilized P-460 moiety of hydroxylamine oxidoreductase, in agreement with the finding by D.J. Miller et al. [J. Gen. Microbiol. 130, 3049-3054 (1984)]. However, cytochrome P-460 had several enzymatic activities which hydroxylamine oxidoreductase showed. Although most of the activities of the cytochrome were lower than the corresponding activities of the oxidoreductase, the hydroxylamine-cytochrome c-552 reductase activity of the cytochrome was about 5-times as high as that of the oxidoreductase.

Nitrosomonas europaea cytochrome P460 is a direct link between nitrification and nitrous oxide emission

“…Recombinant expression and purification of cyt P460 was previously achieved by Elmore et al (29). This method yields a British racing green protein with UV/visible absorption and electron paramagnetic resonance (EPR) spectra consistent with those reported for the enzyme isolated from N. europaea (18,23,30). The absorption spectrum of the as-isolated cyt P460 has a Soret band at 440 nm with a shoulder at 414 nm and Q-band maxima at 570 nm and 627 nm (Fig.…”

“…Steady-state activities of cyt P460 have been reported previously (23,26), but to our knowledge, no studies have presented steady-state activity plots. Fig.…”

“…Steady-state activity assays of cyt P460 were performed under anaerobic conditions using a 2,6-dichlorophenolindophenol (DCPIP)/PMS coupled assay (23) and an N 2 O-selective electrode. By monitoring the decay of the DCPIP absorbance at 605 nm (A 605 ), it was determined that 2 eq of DCPIP was reduced per 1 eq of N 2 O produced under steady-state conditions (SI Appendix, Fig.…”

“…1B). Despite lacking homology to HAO (19,22), the presence of a P460 cofactor in cyt P460 has implicated this enzyme in NH 2 OH oxidation (23)(24)(25).…”

Characterization of the gene encoding hydroxylamine oxidoreductase in Nitrosomonas europaea

“…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).…”