Dataset Information

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

ABSTRACT: 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 (?S‡) 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.

SUBMITTER: Vilbert AC

PROVIDER: S-EPMC5872139 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Vilbert Avery C AC Caranto Jonathan D JD Lancaster Kyle M KM

Chemical science 20171107 2

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 o ...[more]

PMID: 29629106

Similar Datasets

Project description: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.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets