Activation of constitutive nitric oxide synthases by oxidized calmodulin mutants.
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.
Montgomery
Heather J
HJ
Department of Chemistry, University of Waterloo, Waterloo, Ontario N2L 3G1, Canada.
Bartlett
Ryan
R
Perdicakis
Basil
B
Jervis
Eric
E
Squier
Thomas C
TC
Guillemette
J Guy
JG
D000595:Amino Acid Sequence; D000818:Animals; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism*; D002645:Chickens / Q000378:metabolism; D004789:Enzyme Activation / Q000502:physiology; D008715:Methionine / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D010084:Oxidation-Reduction
D002147:Calmodulin; D008715:Methionine; D019001:Nitric Oxide Synthase