Mossbauer, EPR, and optical studies of the P-460 center of hydroxylamine oxidoreductase from Nitrosomonas. A ferrous heme with an unusually large quadrupole splitting

Hydroxylamine oxidoreductase from Nitrosomonas europeae catalyzes the oxidative conversion of NH₂OH to NO₂^-. The enzyme, M(r) = 220,000, has an (αβ)₃ subunit structure with each αβ subunit containing 7-8 c-type hemes and one unusual prosthetic group, termed P-460. The P-460 is also found in a M(r) ~ 17,000 protein (P-460 fragment). Mossbauer spectra of the reduced P-460 groups, in hydroxylamine oxidoreductase and the fragment, exhibit nearly identical quadrupole doublets with an unusually large splitting, ΔE(Q) = 4.21 mm/s (no ferrous heme protein is known with ΔE(Q) > 2.75 mm/s). The observed isomer shift, δ = 0.96 mm/s at 4.2 K, shows that the P-460 iron is high spin ferrous. Treatment of oxidized hydroxylamine oxidoreductase with H₂O₂ followed by reduction or exposure of the native sample to CO led to the disappearance of both the characteristic 460 nm absorption band (ε = 89 mM^-1 cm^-1) and the ΔE(Q) = 4.21 mm/s doublet. The iron of the oxidized P-460 fragment is high spin ferric, with Mossbauer and EPR parameters very similar to those of metmyoglobin. Optical spectra of the reduced P-460 fragment show long wavelength bands at 650 and 688 nm which are sensitive to treatment of the fragment with reagents which react with P-460. These bands were, however, not detected in hydroxylamine oxidoreductase. The spectroscopic and chemical evidence obtained to date suggests strongly that the P-460 iron resides in a heme-like macrocycle although the presumed porphyrin must have some unusual features.