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Activation of constitutive nitric oxide synthases by oxidized calmodulin mutants
Heather J Montgomery1, Ryan Bartlett, Basil Perdicakis
1Department of Chemistry, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada.
Abstract:
Several calmodulin (CaM) mutants were engineered in an effort to identify the functional implications of the oxidation of individual methionines in CaM on the activity of the constitutive isoforms of nitric oxide synthase (NOS). Site-directed mutagenesis was used to substitute the majority of methionines with leucines. Substitution of all nine methionine residues in CaM with leucines had minimal effects on the binding affinity or maximal enzyme activation for either the neuronal (nNOS) or endothelial (eNOS) isoform. Selective substitution permitted determination of the functional consequences of the site-specific oxidation of Met(144) and Met(145) on the regulation of electron transfer within nNOS and eNOS. Site-specific oxidation of Met(144) and Met(145) resulted in changes in the CaM concentration necessary for half-maximal activation of nNOS and eNOS, suggesting that these side chains are involved in stabilizing the productive association between CaM and NOS. However, the site-specific oxidation of Met(144) and Met(145) had essentially no effect on the maximal extent of eNOS activation in the presence of saturating concentrations of CaM. In contrast, the site-specific oxidation of Met(144) (but not Met(145)) resulted in a reduction in the level of nNOS activation that was associated with decreased rates of electron transfer within the reductase domain. Thus, nNOS and eNOS exhibit different functional sensitivities to conditions of oxidative stress that are expected to oxidize CaM. This may underlie some aspects of the observed differences in the sensitivities of proteins in vasculature and neuronal tissues to nitration that are linked to NOS activation and the associated generation of peroxynitrite.
Insights
Oxidative stress impacts calmodulin (CaM) differently in neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS). CaM methionine oxidation affects nNOS more than eNOS, influencing enzyme activation and electron transfer rates.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Calmodulin (CaM) is a crucial calcium-binding protein that regulates various enzymes, including nitric oxide synthases (NOS).
- Oxidative stress can lead to the oxidation of methionine residues in CaM, potentially altering its function.
- Nitric oxide synthases (NOS), particularly neuronal (nNOS) and endothelial (eNOS) isoforms, are critical for physiological processes, and their activity is modulated by CaM.
Purpose of the Study:
- To investigate the functional consequences of methionine oxidation in CaM on the activity of nNOS and eNOS.
- To determine the specific roles of individual methionine residues, particularly Met(144) and Met(145), in CaM-NOS interactions and enzyme regulation.
- To elucidate differential sensitivities of nNOS and eNOS to CaM oxidation under oxidative stress conditions.
Main Methods:
- Engineering of CaM mutants using site-directed mutagenesis, primarily substituting methionine residues with leucines.
- Assessing the effects of CaM mutations on binding affinity and enzyme activation of nNOS and eNOS.
- Analyzing the functional consequences of site-specific methionine oxidation on CaM-NOS complex formation and electron transfer rates within the NOS enzyme.
Main Results:
- Substitution of all nine methionine residues in CaM showed minimal impact on nNOS and eNOS binding or maximal activation.
- Site-specific oxidation of Met(144) and Met(145) altered the CaM concentration required for half-maximal activation of both nNOS and eNOS.
- Oxidation of Met(144) and Met(145) did not affect maximal eNOS activation but reduced nNOS activation and electron transfer rates, with Met(144) oxidation being particularly impactful on nNOS.
Conclusions:
- nNOS and eNOS exhibit distinct functional responses to CaM oxidation, indicating differential sensitivity to oxidative stress.
- The specific methionine residues Met(144) and Met(145) in CaM play roles in stabilizing the CaM-NOS interaction, particularly for nNOS.
- These findings may explain variations in the susceptibility of vascular and neuronal tissues to nitration-induced damage linked to NOS activity and peroxynitrite generation.