Ammonia-oxidizing bacteria (AOB) convert ammonia (NH) to nitrite (NO) as their primary metabolism and thus provide a blueprint for the use of NH as a chemical fuel. The first energy-producing step involves the homotrimeric enzyme hydroxylamine oxidoreductase (HAO), which was originally reported to oxidize hydroxylamine (NHOH) to NO. HAO uses the heme P460 cofactor as the site of catalysis. This heme is supported by seven other hemes in each monomer that mediate electron transfer. Heme P460 cofactors are -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 , which expresses both HAO and cyt P460, these enzymes achieve the oxidation of NHOH and were both originally reported to produce NO. 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 NHOH oxidation. Under aerobic conditions substoichiometric production of NO was observed along with production of nitrous oxide (NO). Under anaerobic conditions, however, NO was the exclusive product of NHOH 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 O, thus producing NO abiotically. Because NO was the true product of one P460 cofactor-containing enzyme, this prompted us to reinvestigate whether NO is enzymatically generated from HAO catalysis. Like cyt P460, we showed that HAO does not produce NO 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 sp. AL212 showed that this enzyme was unable to oxidize NHOH because it lacked a glutamate residue in its secondary coordination sphere that was present in the active cyt P460 variant. Restoring the Glu residue imbued activity, revealing that a second-sphere base is Nature's key to controlled oxidation of NHOH. 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.
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http://dx.doi.org/10.1021/acs.accounts.0c00573 | DOI Listing |
Water Res
June 2024
State Key Laboratory of Urban Water Resource and Environment, School of Environment, Harbin Institute of Technology, Harbin 150090, China. Electronic address:
'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.
View Article and Find Full Text PDFProduct selectivity of ammonia oxidation by ammonia-oxidizing bacteria (AOB) is tightly controlled by metalloenzymes. Hydroxylamine oxidoreductase (HAO) is responsible for the oxidation of hydroxylamine (NHOH) to nitric oxide (NO). The non-metabolic enzyme cytochrome (cyt) P460 also oxidizes NHOH, but instead produces nitrous oxide (NO).
View Article and Find Full Text PDFJ Am Chem Soc
July 2023
Department of Chemistry and Chemical Biology, Baker Laboratory, Cornell University, 162 Sciences Drive, Ithaca, New York 14853, United States.
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 cytochrome P460 may be isolated as a cross-link-deficient proenzyme following anaerobic overexpression in .
View Article and Find Full Text PDFJ Biol Chem
June 2023
School of Life Sciences, University of Essex, Colchester, Essex, United Kingdom; Diamond Light Source Ltd, Harwell Science and Innovation Campus, Didcot, United Kingdom. Electronic address:
The structural basis by which gas-binding heme proteins control their interactions with NO, CO, and O is fundamental to enzymology, biotechnology, and human health. Cytochromes c' (cyts c') are a group of putative NO-binding heme proteins that fall into two families: the well-characterized four alpha helix bundle fold (cyts c'-α) and an unrelated family with a large beta-sheet fold (cyts c'-β) resembling that of cytochromes P460. A recent structure of cyt c'-β from Methylococcus capsulatus Bath revealed two heme pocket phenylalanine residues (Phe 32 and Phe 61) positioned near the distal gas-binding site.
View Article and Find Full Text PDFBiochemistry
April 2022
Division of Physical Sciences, Chemistry, University of Washington-Bothell, Bothell, Washington 98011, United States.
The ammonia-oxidizing bacterium expresses two cytochromes in the P460 superfamily that are predicted to be structurally similar. In one, cytochrome (cyt) P460, the substrate hydroxylamine (NHOH) is converted to nitric oxide (NO) and nitrous oxide (NO) 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 HO forming a ferryl species similar to compound II of peroxidases.
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