Direct Evidence for Nitric Oxide Production by a Nitric-oxide Synthase-like Protein from Bacillus subtilis
Open accessAbstract
Nitric-oxide synthases (NOSs) are widely distributed among prokaryotes and eukaryotes and have diverse functions in physiology. Recent genome sequencing revealed NOS-like protein in bacteria, but whether these proteins generate nitric oxide is unknown. We therefore cloned, expressed, and purified a NOS-like protein from Bacillus subtilis (bsNOS) and characterized its catalytic parameters in both multiple and single turnover reactions. bsNOS was dimeric, bound l-Arg and 6R-tetrahydrobiopterin with similar affinity as mammalian NOS, and generated nitrite from l-Arg when incubated with NADPH and a mammalian NOS reductase domain. Stopped-flow analysis showed that ferrous bsNOS reacted with O(2) to form a transient heme Fe(II)O(2) species in the presence of either Arg or the reaction intermediate N-hydroxy-l-arginine. In the latter case, disappearance of the Fe(II)O(2) species was kinetically and quantitatively coupled to formation of a transient heme Fe(III)NO product, which then dissociated to form ferric bsNOS. This behavior mirrors mammalian NOS enzymes and unambiguously shows that bsNOS can generate NO. NO formation required a bound tetrahydropteridine, and the kinetic effects of this cofactor were consistent with it donating an electron to the Fe(II)O(2) intermediate during the reaction. Dissociation of the heme Fe(III)NO product was much slower in bsNOS than in mammalian NOS. This constrains allowable rates of ferric heme reduction by a protein redox partner and underscores the utility of using a tetrahydropteridine electron donor in bsNOS.
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iNOS and nNOS show higher affinity toward BH4 than for BH2 (around 10-fold in the absence of l-arginine).
“iNOS and nNOS show higher affinity toward BH4 than for BH2 (around 10-fold in the absence of l-arginine) (6, 514, 753); however, eNOS shows almost identical binding affinities for BH4 and BH2, and it is thus very sensitive to imbalances in the concentrations of BH4 and BH2 (190).”
From Sources of Vascular Nitric Oxide and Reactive Oxygen Species and Their Regulation (2019) · cites this paper as [6]
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- Authors
- Adak, S., Aulak, K. S., & Stuehr, D. J.
- Affiliations
- Cleveland Clinic (US)
- Corresponding author
- Adak, Subrata
- Stuehr, Dennis J.
- Venue
- Journal of Biological Chemistry
- Publisher
- Elsevier BV
- Published
- 2002-05
- Volume
- 277
- Issue
- 18
- Pages
- 16167-16171
- ISSN
- 0021-9258 (ISSN-L)
- OpenAlex
- W2077437981
- Cited by
- 195
- References
- 24
- Citations by year
- 2026: 4 · 2025: 6 · 2024: 4 · 2022: 10 · 2021: 8 · 2020: 3 · 2019: 4 · 2018: 10 · 2017: 7 · 2016: 11 · 2015: 6 · 2014: 7 · 2013: 5 · 2012: 5
- FWCI
- 7.84
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- Locations
- Journal of Biological Chemistry (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Funders
- National Cancer Institute (CA-53914)
- Topics
- Physical Sciences › Chemistry › Inorganic Chemistry › Metal-Catalyzed Oxygenation Mechanisms
- Health Sciences › Medicine › Physiology › Nitric Oxide and Endothelin Effects
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Hemoglobin structure and function
- Keywords
- nitric oxide synthase, Bacillus subtilis, nitric oxide production, tetrahydrobiopterin, heme iron oxidation, L-arginine, NADPH, enzyme kinetics, protein dimerization
- MeSH terms
- Amino Acid Sequence, Animals, Arginine, Bacillus subtilis, Bacterial Proteins, Biopterins, Catalysis, Kinetics, Mammals, Molecular Sequence Data, Nitric Oxide, Sequence Alignment, Sequence Homology, Amino Acid, Protein Structure, Secondary, Nitric Oxide Synthase, Dimerization
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