Cyt P460 Research Articles

Nitrate-dependent anaerobic methane oxidation coupled to Fe(III) reduction as a source of ammonium and nitrous oxide

‘Candidatus Methanoperedens nitroreducens’ is an archaeal methanotroph with global importance that links carbon and nitrogen cycles and great potential for sustainable operation of wastewater treatment. It has been reported to mediate the anaerobic oxidation of methane through a reverse methanogenesis pathway while reducing nitrate to nitrite. Here, we demonstrate that ‘Ca. M. nitroreducens’ reduces ferric iron forming ammonium (23.1 %) and nitrous oxide (N2O, 46.5 %) from nitrate. These results are supported with the upregulation of genes coding for proteins responsible for dissimilatory nitrate reduction to ammonium (nrfA), N2O formation (norV, cyt P460), and multiple multiheme c-type cytochromes for ferric iron reduction. Concomitantly, an increase in the N2O-reducing SJA-28 lineage and a decrease in the nitrite-reducing ‘Candidatus Methylomirabilis oxyfera’ are consistent with the changes in ‘Ca. M. nitroreducens’ end products. These findings demonstrate the highly flexible physiology of ‘Ca. M. nitroreducens’ in anaerobic ecosystems with diverse electron acceptor conditions, and further reveals its roles in linking methane oxidation to global biogeochemical cycles. ‘Ca. M. nitroreducens’ could significantly affect the bioavailability of nitrogen sources as well as the emission of greenhouse gas in natural ecosystems and wastewater treatment plants.

Cytochrome P460 Cofactor Maturation Proceeds via Peroxide-Dependent Post-translational Modification.

Cytochrome P460s are heme enzymes that oxidize hydroxylamine to nitrous oxide. They bear specialized "heme P460" cofactors that are cross-linked to their host polypeptides by a post-translationally modified lysine residue. Wild-type N. europaea cytochrome P460 may be isolated as a cross-link-deficient proenzyme following anaerobic overexpression in E. coli. When treated with peroxide, this proenzyme undergoes maturation to active enzyme with spectroscopic and catalytic properties that match wild-type cyt P460. This maturation reactivity requires no chaperones─it is intrinsic to the protein. This behavior extends to the broader cytochrome c'β superfamily. Accumulated data reveal key contributions from the secondary coordination sphere that enable selective, complete maturation. Spectroscopic data support the intermediacy of a ferryl species along the maturation pathway.

Outer coordination sphere influences on cofactor maturation and substrate oxidation by cytochrome P460.

Product selectivity of ammonia oxidation by ammonia-oxidizing bacteria (AOB) is tightly controlled by metalloenzymes. Hydroxylamine oxidoreductase (HAO) is responsible for the oxidation of hydroxylamine (NH2OH) to nitric oxide (NO). The non-metabolic enzyme cytochrome (cyt) P460 also oxidizes NH2OH, but instead produces nitrous oxide (N2O). While both enzymes use a heme P460 cofactor, they selectively oxidize NH2OH to different products. Previously reported structures of Nitrosomonas sp. AL212 cyt P460 show that a capping phenylalanine residue rotates upon ligand binding, suggesting that this Phe may influence substrate and/or product binding. Here, we show via substitutions of the capping Phe in Nitrosomonas europaea cyt P460 that the bulky phenyl side-chain promotes the heme-lysine cross-link forming reaction operative in maturing the cofactor. Additionally, the Phe side-chain plays an important role in modulating product selectivity between N2O and NO during NH2OH oxidation under aerobic conditions. A picture emerges where the sterics and electrostatics of the side-chain in this capping position control the kinetics of N2O formation and NO binding affinity. This demonstrates how the outer coordination sphere of cyt P460 is tuned not only for selective NH2OH oxidation, but also for the autocatalytic cross-link forming reaction that imbues activity to an otherwise inactive protein.

Structural Characterization of Cytochrome c′β‐Met from an Ammonia-Oxidizing Bacterium

The ammonia-oxidizing bacterium Nitrosomonas europaea expresses two cytochromes in the P460 superfamily that are predicted to be structurally similar. In one, cytochrome (cyt) P460, the substrate hydroxylamine (NH2OH) is converted to nitric oxide (NO) and nitrous oxide (N2O) requiring a unique heme-lysyl cross-link in the catalytic cofactor. In the second, cyt , the cross-link is absent, and the cytochrome instead binds H2O2 forming a ferryl species similar to compound II of peroxidases. Here, we report the 1.80 Å crystal structure of cyt ─a well-expressed protein in N. europaea with a lysine to a methionine replacement at the cross-linking position. The structure of cyt is characterized by a large β-sheet typical of P460 members; however, several localized structural differences render cyt distinct. This includes a large lasso-like loop at the "top" of the cytochrome that is not observed in other structurally characterized members. Active site variation is also observed, especially in comparison to its closest homologue cyt from the methane-oxidizing Methylococcus capsulatus Bath, which also lacks the cross-link. The phenylalanine "cap" which is presumed to control small ligand access to the distal heme iron is replaced with an arginine, reminiscent of the strictly conserved distal arginine in peroxidases and to the NH2OH-oxidizing cytochromes P460. A critical proton-transferring glutamate residue required for NH2OH oxidation is nevertheless missing in the active site. This in part explains the inability of cyt to oxidize NH2OH. Our structure also rationalizes the absence of a methionyl cross-link, although the side chain's spatial position in the structure does not eliminate the possibility that it could form under certain conditions.

Heme P460: A (Cross) Link to Nitric Oxide.

Ammonia-oxidizing bacteria (AOB) convert ammonia (NH3) to nitrite (NO2-) as their primary metabolism and thus provide a blueprint for the use of NH3 as a chemical fuel. The first energy-producing step involves the homotrimeric enzyme hydroxylamine oxidoreductase (HAO), which was originally reported to oxidize hydroxylamine (NH2OH) to NO2-. HAO uses the heme P460 cofactor as the site of catalysis. This heme is supported by seven other c hemes in each monomer that mediate electron transfer. Heme P460 cofactors are c-heme-based cofactors that have atypical protein cross-links between the peptide backbone and the porphyrin macrocycle. This cofactor has been observed in both the HAO and cytochrome (cyt) P460 protein families. However, there are differences; specifically, HAO uses a single tyrosine residue to form two covalent attachments to the macrocycle whereas cyt P460 uses a lysine residue to form one. In Nitrosomonas europaea, which expresses both HAO and cyt P460, these enzymes achieve the oxidation of NH2OH and were both originally reported to produce NO2-. Each can inspire means to effect controlled release of chemical energy.Spectroscopically studying the P460 cofactors of HAO is complicated by the 21 non-P460 heme cofactors, which obscure the active site. However, monoheme cyt P460 is more approachable biochemically and spectroscopically. Thus, we have used cyt P460 to study biological NH2OH oxidation. Under aerobic conditions substoichiometric production of NO2- was observed along with production of nitrous oxide (N2O). Under anaerobic conditions, however, N2O was the exclusive product of NH2OH oxidation. We have advanced our understanding of the mechanism of this enzyme and have showed that a key intermediate is a ferric nitrosyl that can dissociate the bound nitric oxide (NO) molecule and react with O2, thus producing NO2- abiotically. Because N2O was the true product of one P460 cofactor-containing enzyme, this prompted us to reinvestigate whether NO2- is enzymatically generated from HAO catalysis. Like cyt P460, we showed that HAO does not produce NO2- enzymatically, but unlike cyt P460, its final product is NO, establishing it as an intermediate of nitrification. More broadly, NO can be recognized as a molecule common to the primary metabolisms of all organisms involved in nitrogen "defixation".Delving deeper into cyt P460 yielded insights broadly applicable to controlled biochemical redox processes. Studies of an inactive cyt P460 from Nitrosomonas sp. AL212 showed that this enzyme was unable to oxidize NH2OH because it lacked a glutamate residue in its secondary coordination sphere that was present in the active N. europaea cyt P460 variant. Restoring the Glu residue imbued activity, revealing that a second-sphere base is Nature's key to controlled oxidation of NH2OH. A key lesson of bioinorganic chemistry is reinforced: the polypeptide matrix is an essential part of dictating function. Our work also exposed some key functional contributions of noncanonical heme-protein cross-links. The heme-Lys cross-link of cyt P460 enforces the relative position of the cofactor and second-sphere residues. Moreover, the cross-link prevents the dissociation of the axial histidine residue, which stops catalysis, emphasizing the importance of this unique post-translational modification.

The Heme-Lys Cross-Link in Cytochrome P460 Promotes Catalysis by Enforcing Secondary Coordination Sphere Architecture.

Cytochrome (cyt) P460 is a c-type monoheme enzyme found in ammonia-oxidizing bacteria (AOB) and methanotrophs; additionally, genes encoding it have been found in some pathogenic bacteria. Cyt P460 is defined by a unique post-translational modification to the heme macrocycle, where a lysine (Lys) residue covalently attaches to the 13' meso carbon of the porphyrin, modifying this heme macrocycle into the enzyme's eponymous P460 cofactor, similar to the cofactor found in the enzyme hydroxylamine oxidoreductase. This cross-link imbues the protein with unique spectroscopic properties, the most obvious of which is the enzyme's green color in solution. Cyt P460 from the AOB Nitrosomonas europaea is a homodimeric redox enzyme that produces nitrous oxide (N2O) from 2 equiv of hydroxylamine. Mutation of the Lys cross-link results in spectroscopic features that are more similar to those of standard cyt c' proteins and renders the enzyme catalytically incompetent for NH2OH oxidation. Recently, the necessity of a second-sphere glutamate (Glu) residue for redox catalysis was established; it plausibly serves as proton relay during the first oxidative half of the catalytic cycle. Herein, we report the first crystal structure of a cross-link deficient cyt P460. This structure shows that the positioning of the catalytically essential Glu changes by approximately 0.8 Å when compared to a cross-linked, catalytically competent cyt P460. It appears that the heme-Lys cross-link affects the relative position of the P460 cofactor with respect to the second-sphere Glu residue, therefore dictating the catalytic competency of the enzyme.

Cytochrome c'β-Met Is a Variant in the P460 Superfamily Lacking the Heme-Lysyl Cross-Link: A Peroxidase Mimic Generating a Ferryl Intermediate.

A defining characteristic of bacterial cytochromes (cyt's) in the P460 family is an unusual cross-link connecting the heme porphyrin to the side chain of a lysyl residue in the protein backbone. Here, via proteomics of the periplasmic fraction of the ammonia-oxidizing bacterium (AOB) Nitrosomonas europaea, we report the identification of a variant member of the P460 family that contains a methionyl residue in place of the cross-linking lysine. We formally designate this protein cytochrome "c'β-Met" to distinguish it from other members bearing different residues at this position (e.g., cyt c'β-Phe from the methane-oxidizing Methylococcus capsulatus Bath). As isolated, the monoheme cyt c'β-Met is high-spin (S = 5/2). Optical spectroscopy suggests that a cross-link is absent. Hydroxylamine, the substrate for the cross-linked cyt P460 from N. europaea, did not appreciably alter the optical spectrum of cyt c'β with up to 1000-fold excess at pH 7.5. Cyt c'β-Met did however bind 1 equiv of H2O2, and with a slight excess, Mössbauer spectroscopy indicated the formation of a semistable ferryl (FeIV═O) Compound II-like species. The corresponding electron paramagnetic resonance showed a very low intensity signal indicative of a radical at g = 2.0. Furthermore, cyt c'β-Met exhibited guaiacol-dependent peroxidase activity (kcat = 20.0 ± 1.2 s-1; KM = 2.6 ± 0.4 mM). Unlike cyt c'β-Met, cyt P460 showed evidence of heme inactivation in the presence of 2 equiv of H2O2 with no appreciable guaiacol-dependent peroxidase activity. Mutagenesis of the cross-linking lysyl residue to an alanine in cyt P460, however, reversed this lack of activity.

Influences of the heme-lysine crosslink in cytochrome P460 over redox catalysis and nitric oxide sensitivity.

Ammonia (NH3)-oxidizing bacteria (AOB) derive total energy for life from the multi-electron oxidation of NH3 to nitrite (NO2-). One obligate intermediate of this metabolism is hydroxylamine (NH2OH), which can be oxidized to the potent greenhouse agent nitrous oxide (N2O) by the AOB enzyme cytochrome (cyt) P460. We have now spectroscopically characterized a 6-coordinate (6c) {FeNO}7 intermediate on the NH2OH oxidation pathway of cyt P460. This species has two fates: it can either be oxidized to the {FeNO}6 that then undergoes attack by NH2OH to ultimately generate N2O, or it can lose its axial His ligand, thus generating a stable, off-pathway 5-coordinate (5c) {FeNO}7 species. We show that the wild type (WT) cyt P460 exhibits a slow nitric oxide (NO)-independent conversion (kHis-off = 2.90 × 10-3 s-1), whereas a cross-link-deficient Lys70Tyr cyt P460 mutant protein underwent His dissociation via both a NO-independent (kHis-off = 3.8 × 10-4 s-1) and a NO-dependent pathway [kHis-off(NO) = 790 M-1 s-1]. Eyring analyses of the NO-independent pathways for these two proteins revealed a significantly larger (ca. 27 cal mol-1 K-1) activation entropy (ΔS‡) in the cross-link-deficient mutant. Our results suggest that the Lys-heme cross-link confers rigidity to the positioning of the heme P460 cofactor to avoid the fast NO-dependent His dissociation pathway and subsequent formation of the off-pathway 5c {FeNO}7 species. The relevance of these findings to NO signaling proteins such as heme-nitric oxide/oxygen binding (H-NOX) is also discussed.

The Eponymous Cofactors in Cytochrome P460s from Ammonia-Oxidizing Bacteria Are Iron Porphyrinoids Whose Macrocycles Are Dibasic.

The enzymes hydroxylamine oxidoreductase and cytochrome (cyt) P460 contain related unconventional "heme P460" cofactors. These cofactors are unusual in their inclusion of nonstandard cross-links between amino acid side chains and the heme macrocycle. Mutagenesis studies performed on the Nitrosomonas europaea cyt P460 that remove its lysine-heme cross-link show that the cross-link is key to defining the spectroscopic properties and kinetic competence of the enzyme. However, exactly how this cross-link confers these features remains unclear. Here we report the 1.45 Å crystal structure of cyt P460 from Nitrosomonas sp. AL212 and conclude that the cross-link does not lead to a change in hybridization of the heme carbon participating in the cross-link but rather enforces structural distortions to the macrocycle away from planarity. Time-dependent density functional theory coupled to experimental structural and spectroscopic analysis suggest that this geometric distortion is sufficient to define the spectroscopic properties of the heme P460 cofactor and provide clues toward establishing a relationship between heme P460 electronic structure and function.

Nitrosomonas europaea cytochrome P460 is a direct link between nitrification and nitrous oxide emission

Ammonia oxidizing bacteria (AOB) are major contributors to the emission of nitrous oxide (N2O). It has been proposed that N2O is produced by reduction of NO. Here, we report that the enzyme cytochrome (cyt) P460 from the AOB Nitrosomonas europaea converts hydroxylamine (NH2OH) quantitatively to N2O under anaerobic conditions. Previous literature reported that this enzyme oxidizes NH2OH to nitrite ([Formula: see text]) under aerobic conditions. Although we observe [Formula: see text] formation under aerobic conditions, its concentration is not stoichiometric with the NH2OH concentration. By contrast, under anaerobic conditions, the enzyme uses 4 oxidizing equivalents (eq) to convert 2 eq of NH2OH to N2O. Enzyme kinetics coupled to UV/visible absorption and electron paramagnetic resonance (EPR) spectroscopies support a mechanism in which an FeIII-NH2OH adduct of cyt P460 is oxidized to an {FeNO}6 unit. This species subsequently undergoes nucleophilic attack by a second equivalent of NH2OH, forming the N-N bond of N2O during a bimolecular, rate-determining step. We propose that [Formula: see text] results when nitric oxide (NO) dissociates from the {FeNO}6 intermediate and reacts with dioxygen. Thus, [Formula: see text] is not a direct product of cyt P460 activity. We hypothesize that the cyt P460 oxidation of NH2OH contributes to NO and N2O emissions from nitrifying microorganisms.