Resolution of Multiple Heme Centers of Hydroxylamine Oxidoreductase from Nitrosomonas. 1. Electron Paramagnetic Resonance Spectroscopy

John D. Lipscomb, Alan B. Hooper

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24 Scopus citations


Hydroxylamine oxidoreductase (HAO) from Nitrosomonas europeae [M, 220000, subunit structure of (aβ)3 with seven c-type hemes and one P-460-type heme per ß subunit] catalyzes the oxidative conversion of NH2OH to NO2”. We have used electron paramagnetic resonance (EPR) spectroscopy to monitor a reductive titration of the enzyme. The spectra show that the c-type hemes can be placed into at least four groups with different oxidation-reduction potentials and protein environments. Since the hemes are reduced sequentially, g-value assignments can be made for three of the major species (g = 3.38, 1.95, 0.7; g = 3.06, 2.14, 1.35; and g = 2.98, 2.24, 1.44), and the spectrum of each can be isolated from the composite spectrum of the protein. Quantitation of these three spectra suggests that approximately one-third of the heme in the enzyme resides in other species. Some of the unquantitated heme may contribute to one or more novel EPR active species with g values at g = 2.7, g = 1.85, and g = 1.66. A second fraction of this heme is probably EPR silent since high- and low-spin heme signals absent from the spectrum of resting HAO appear during the final half of the reductive titration. One of these newly EPR active species, with characteristic high-spin heme g values at 6.45 and 5.6, reduces concomitantly with the appearance of the optical spectrum of the reduced P-460 heme. On this basis, P-460 is tentatively assigned as the high-spin heme. Since no high-spin heme signal is observed in the resting enzyme, P-460 must either undergo a spin conversion as the other hemes are reduced or be EPR silent due to spin coupling or fast electronic spin relaxation. EPR spectra also show that NH2OH reduces approximately 45% of the hemes when complexed anaerobically with HAO. A new low-spin heme (g = 2.86, 2.3) becomes EPR active in this complex, and the g values of most of the other EPR active hemes shift slightly.

Original languageEnglish (US)
Pages (from-to)3965-3972
Number of pages8
Issue number17
StatePublished - Aug 1982

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