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Title: 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 (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 tomore » 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.« less

Authors:
 [1];  [1]; ORCiD logo [1]
  1. Department of Chemistry and Chemical Biology, Baker Laboratory, Cornell University, Ithaca, USA
Publication Date:
Research Org.:
Cornell Univ., Ithaca, NY (United States); Stanford Univ., CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES)
OSTI Identifier:
1415513
Alternate Identifier(s):
OSTI ID: 1505565
Grant/Contract Number:  
AC02-76SF00515; SC0013997
Resource Type:
Published Article
Journal Name:
Chemical Science
Additional Journal Information:
Journal Name: Chemical Science Journal Volume: 9 Journal Issue: 2; Journal ID: ISSN 2041-6520
Publisher:
Royal Society of Chemistry (RSC)
Country of Publication:
United Kingdom
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY

Citation Formats

Vilbert, Avery C., Caranto, Jonathan D., and Lancaster, Kyle M. Influences of the heme-lysine crosslink in cytochrome P460 over redox catalysis and nitric oxide sensitivity. United Kingdom: N. p., 2018. Web. https://doi.org/10.1039/C7SC03450D.
Vilbert, Avery C., Caranto, Jonathan D., & Lancaster, Kyle M. Influences of the heme-lysine crosslink in cytochrome P460 over redox catalysis and nitric oxide sensitivity. United Kingdom. https://doi.org/10.1039/C7SC03450D
Vilbert, Avery C., Caranto, Jonathan D., and Lancaster, Kyle M. Mon . "Influences of the heme-lysine crosslink in cytochrome P460 over redox catalysis and nitric oxide sensitivity". United Kingdom. https://doi.org/10.1039/C7SC03450D.
@article{osti_1415513,
title = {Influences of the heme-lysine crosslink in cytochrome P460 over redox catalysis and nitric oxide sensitivity},
author = {Vilbert, Avery C. and Caranto, Jonathan D. and Lancaster, Kyle M.},
abstractNote = {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.},
doi = {10.1039/C7SC03450D},
journal = {Chemical Science},
number = 2,
volume = 9,
place = {United Kingdom},
year = {2018},
month = {1}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record
https://doi.org/10.1039/C7SC03450D

Citation Metrics:
Cited by: 7 works
Citation information provided by
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Figures / Tables:

Fig. 1 Fig. 1: Views of the top (a) and side (b) of the Nitrosomonas europaea HAO heme P460 cofactor (2.1 Å resolution X-ray crystal structure, PDBID 4FAS) and the top (c) and side (d) of the N. europaea cytochrome (cyt) P460 heme P460 cofactor (1.8 Å resolution X-ray crystal structure, PDBIDmore » 2JE3). Both cofactors are c-heme cofactors with additional covalent amino acid side chain attachments. In HAO, Tyr467 from a neighboring subunit cross-links via the Cε at the 5' meso carbon of the porphyrin and via the phenolate O at the neighboring pyrrole α-carbon. In cyt P460, the Lys70 amine N cross-links to the 13' meso carbon.« less

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