Unusual spin interactions in the 24 heme hydroxylamine oxidoreductase and diheme cytochrome c 554 from nitrosomonas

Abstract Nitrosomonas oxidizes NH 3 to HNO 2 with NH 2 OH as an intermediate. Oxidation of NH 2 OH appears to involve two multiheme cytochromes: hydroxylamine oxidoreductase (HAO) [1] and cytochrome c 554 [2]. Hemes of HAO have midpoint potentials varying from +100 mV to −350 mV [3]. HAO can accept electrons from NH 2 OH and pass them to cyt c 554 (midpoint potential −50 mV, 2). HAO , with an α 3 β 3 subunit structure, contains 7 c -type hemes and one unique heme P460 per αβ dimer. The CO-binding heme P460 is essential for the NH 2 OH dehydrogenase activity and is specifically destroyed by H 2 O 2 . EPR studies of HAO reveal several classes of low spin (s = 1 2 ) hemes [4]. Two species, accounting for half of the hemes, have been assigned g-values by reductive EPR titration; g = 3.06, 2.14, 1.35 and g = 2.98, 2.24, 1.44 [5]. Only four other EPR signals appear in the oxidized spectrum (g = 3.38, 2.70, 1.85 and 1.66). These resonances titrate coordinately but are not typical of magnetically isolated heme spectra. The apparent g-values of these 4 resonances are frequenCy dependent suggesting that they arise from spin-interactions of the hemes. Frequency dependence of the type observed has not been previously reported. The Mossbauer spectrum of ferric HAO contains a quadrupole doublet at 4.2 K in addition to the expected broad magnetically split spectrum, typical of s = 1 2 hemes. This doublet, which corresponds to at least one and probably two irons per αβ-dimer, has parameters (ΔE Q = 2.1 mm/s and δ Fe = 0.24 mm/s) which are typical of either low spin ferric heme with fast electronic spin relaxation or a pair of spin-coupled hemes [6]. We speculate that this doublet may be associated with the four frequency dependent EPR resonances. Heme P460 is not a component of the latter species since selective destruction of P460 by H 2 O 2 fails to alter the EPR spectrum of the oxidized HAO. Thus heme P460 of native HAO is EPR silent. Cytochrome c554 at pH 7 has an unusual 10 K EPR spectrum (g = 4.18, 3.85) similar to intermediate spin (s = 3 2 ) complexes. At pH 4 the EPR spectrum consists of one high spin (g = 6.0, 2.0 and one low spin (g = 2.93, 2.25, 1.52) component. At pH 2 a single high spin component (g = 6.0, 2.0) is present, whereas two low spin forms are observed at pH 10.5. Optical spectra of oxidized cyt c 554 at 20 °C are consistent with high spin heme at pH 4 and low spin heme at pH 10.5. Reduced cyt c 554 reacts with O 2 and binds CO at pH 4: the CO spectrum has two Soret maxima indicating a different interaction with each heme. 1 H-NMR spectra at room temperature show contact shifted heme methylene resonances in both the low spin (10–30 ppm) and high spin (60–100 ppm) Fe 3+ spectral regions at all pH values between 4.5 and 9. Contact shifted resonances similar to those reported for s = 3 2 model heme complexes are not observed at this temperature. We conclude that the unusual low temperature EPR spectrum at pH 7 results either from a spin conversion or interaction between high and low spin hemes. EPR, NMR and optical spectra show that this is a different type of heme-heme interaction than observed with diheme cyt c ′.