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08:32
Application of Genetically Encoded Fluorescent Nitric Oxide (NO•) Probes, the geNOps, for Real-time Imaging of NO• Signals in Single Cells
Published on: March 16, 2017
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Dissecting structural and electronic effects in inducible nitric oxide synthase.
Luciana Hannibal1, Richard C Page2, Mohammad Mahfuzul Haque1
1*Department of Pathobiology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 44195, U.S.A.
The Biochemical Journal
|January 23, 2015
Summary
Mesohaem substitution in inducible nitric oxide synthase (iNOS) enhances nitric oxide (NO) production by reducing intermediate species formation. This study reveals electronic effects, not structural changes, drive iNOS catalytic improvements.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Nitric oxide synthases (NOSs) are crucial haem-thiolate enzymes.
- Inducible NOS (iNOS) produces nitric oxide (NO) during inflammation.
- Catalytic efficiency of iNOS depends on haem midpoint potential and substrate interactions.
Purpose of the Study:
- To investigate the impact of mesohaem substitution on iNOS catalytic activity.
- To compare native iNOS with iNOS-mesohaem and W188H-mesohaem mutants.
- To elucidate structural and electronic effects of mesohaem substitution on iNOS function.
Main Methods:
- Enzyme characterization of iNOS-mesohaem and W188H-mesohaem.
- Single turnover kinetic assays using L-arginine and N-hydroxy-L-arginine.
- X-ray crystallography of mesohaem-substituted iNOS.
Main Results:
- Mesohaem substitution in iNOS and W188H mutant maintained stability and substrate binding.
- Mesohaem triggered higher rates of Fe(II)O₂ conversion and altered kinetic parameters.
- Mesohaem substitution significantly reduced intermediate species P, enhancing NO synthesis proficiency.
Conclusions:
- Mesohaem substitution in iNOS enhances NO synthesis by modulating electronic properties.
- Structural integrity of iNOS is largely preserved upon mesohaem substitution.
- Observed kinetic improvements are primarily attributed to electronic effects rather than structural alterations.
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