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

Journal Article · · Chemical Science
DOI:https://doi.org/10.1039/C7SC03450D· OSTI ID:1415513
 [1];  [1]; ORCiD logo [1]
  1. Department of Chemistry and Chemical Biology, Baker Laboratory, Cornell University, Ithaca, USA
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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.

Research Organization:
Cornell Univ., Ithaca, NY (United States); Stanford Univ., CA (United States)
Sponsoring Organization:
USDOE Office of Science (SC), Basic Energy Sciences (BES)
Grant/Contract Number:
AC02-76SF00515; SC0013997
OSTI ID:
1415513
Alternate ID(s):
OSTI ID: 1505565
Journal Information:
Chemical Science, Journal Name: Chemical Science Vol. 9 Journal Issue: 2; ISSN 2041-6520
Publisher:
Royal Society of Chemistry (RSC)Copyright Statement
Country of Publication:
United Kingdom
Language:
English
Citation Metrics:
Cited by: 20 works
Citation information provided by
Web of Science

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