Kristoffer K. Andersson

O2 and H2O are each the source of one O in NO− 2 produced from NH3 by Nitrosomonas: 15N-NMR evidence

The exchange of 18O between H2 18O and exogeneously added 15N16O− 2 which occurs during oxidation of ammonia by Nitrosomonas is shown to occur one oxygen at a time. Conditions in which the exchange is diminished (notably the presence of 14NO– 2 and CCCP) allowed demonstration that water and dioxygen are each the source of one oxygen in nitrite produced from 15NH3. The nitrate produced in the presence of 18O2 consisted of 67 and 0% 15N18O16O− and 15N18O18O−, respectively. Analysis was made using the 18O‐isotope shift in 15N‐NMR.

P460 of hydroxylamine oxidoreductase of Nitrosomonas europaea: Soret resonance Raman evidence for a novel heme-like structure.

P460, an iron-containing chromophore at the active site of Hydroxylamine Oxidoreductase of the ammonia-oxidizing bacterium Nitrosomonas europaea, is a macrocycle of unknown structure with a Soret-like 460-nm absorption band in the ferrous form. The pigment can also be isolated in a peptide, P460-Fragment. Resonance Raman spectroscopy (lambda ex = 457.9 nm) suggests that P460 is a new type of heme with symmetry properties lower than those of protophorphyrin IX or chlorins and similar to those of chlorophylls and isobacteriochlorins. Some of the resonance Raman vibrations of P460 are shifted in HAO as compared to those of P460-Fragment.

Diheme cytochrome c-554 from Nitrosomonas: Soret resonance Raman indication of an unusual ferric 5-coordinate structure

The diheme cytochrome c‐554 which participates in ammonia oxidation in the chemoautotroph, Nitrosomonas europaea has been studied by Soret excitation resonance Raman spectroscopy. The Raman spectrum of reduced cytochrome c‐554 at neutral pH is similar classical 6‐coordinate low‐spin ferrous mammalian cytochrome c. In contrast, the spectrum of ferric cytochrome c‐554 suggests a 5‐coordinate state which is unusual for c hemes. The oxidized spectrum closely resembles that of horseradish peroxidase (HRP) or cytochrome c peroxidase (CcP) at pH 6.4. The narrow linewidth of the heme core‐size vibrations indicates that both heme irons of c‐554 have similar geometries.

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 ′.

Oxygenation by Methane Monooxygenase: Oxygen Activation and Component Interactions

Methanotrophic bacteria possess the unique ability to utilize methane as the sole source of carbon and energy. Indeed, methane is the only carbon source capable of sustaining vigorous and long term growth of these organisms1. The methanolytic activity of methanotrophs can be ascribed to the elaboration of a unique enzyme, methane monooxygenase2 (MMO), which catalyzes the following reaction: n n

Methane monooxygenase component B and reductase alter the regioselectivity of the hydroxylase component-catalyzed reactions. A novel role for protein-protein interactions in an oxygenase mechanism.

Methane + {O_2} + NADH + {H^ + } to NA{D^ + } + {H_2}O + Methanol

Heme P460 of Hydroxylamine Oxidoreductase of Nitrosomonas

Abstract Tetraheme cytochrome c-544 is a part of the ammonia oxidizing system in Nitrosomonas europaea. The magnetic susceptibility of the neutral pH form of oxidized cytochrome c-554 has been studied over the temperature range 12–180 K. The experimental data are consistent with a composition of 19–24% (20–27%) of high-spin Fe(III) with the zero-field splitting parameter, D, in the range 12 ± 2 c−1 (−15.5 ± 4.5 cm−1) and 81–76% (80–73%) of the low-spin Fe(III) prototype species previously suggested from Mossbauer studies. 1H-NMR spectra at ambient temperature show significant contributions from one high-spin and at least three different low-spin Fe(III) species. Our data are not in agreement with a homogeneous heme-iron population in a 5-coordinated out-of-plane conformation, as suggested on the basis of previous resonance Raman data.