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

K. K. Andersson, T. A. Kent, J. D. Lipscomb

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)6833-6840
Number of pages8
JournalJournal of Biological Chemistry
Volume259
Issue number11
StatePublished - Jan 1 1984

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hydroxylamine dehydrogenase
Nitrosomonas
Heme
Paramagnetic resonance
Iron
Metmyoglobin
Hemeproteins
Porphyrins
Carbon Monoxide
Prosthetics
Isomers
Absorption spectra
Wavelength
Enzymes

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@article{614ac6e09bb042188cfc3639fe0e553c,
title = "Mossbauer, EPR, and optical studies of the P-460 center of hydroxylamine oxidoreductase from Nitrosomonas. A ferrous heme with an unusually large quadrupole splitting",
abstract = "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.",
author = "Andersson, {K. K.} and Kent, {T. A.} and Lipscomb, {J. D.}",
year = "1984",
month = "1",
day = "1",
language = "English (US)",
volume = "259",
pages = "6833--6840",
journal = "Journal of Biological Chemistry",
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TY - JOUR

T1 - Mossbauer, EPR, and optical studies of the P-460 center of hydroxylamine oxidoreductase from Nitrosomonas. A ferrous heme with an unusually large quadrupole splitting

AU - Andersson, K. K.

AU - Kent, T. A.

AU - Lipscomb, J. D.

PY - 1984/1/1

Y1 - 1984/1/1

N2 - 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.

AB - 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.

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