Three nitric oxide synthase isoforms (NOSs) have evolved to function broadly in human disease. Our overall goal is to understand how NO synthesis occurs and is controlled at a molecular level in each isoform. Our work with neuronal NOS led to a model that has calmodulin controlling electron transfer between the NOS flavin-containing reductase domain and a heme group located in the NOS oxygenase domain, which enables the heme iron to bind and activate 02 and catalyze stepwise NO synthesis from L-arginine. We propose that NO synthesis in this system is controlled by four related features that are distinct from NO biosynthetic steps: (1) Flavin and heme iron reduction, (2) Calmodulin activation of the NOS reductase domain, (3) Relative heme iron affinity toward 02 versus NO, and (4) Formation of an enzyme heme-NO complex. Moreover, we believe that each NOS isoform may differ in how these four features contribute to regulate its activity. We will utilize biochemical, kinetic, molecular biological, and biophysical methods to closely examine the four features in each NOS isoform:
Aim I. Compare rates of flavin and heme iron reduction in each NOS, flavin and heme iron reduction potentials, and substrat effects on these parameters.
Aim H. Utilize calmodulin-troponin c chimeras to investigate how calmodulin activates the reductase domain of each NOS.
Aim III Determine the kinetics of 02 and NO binding in each NOS, and substrate effects Aim IV. Investigate the basis for heme-NO complex formation in each NOS and whether NO complex formation modulates NOS 02 response.
Aim V. Critically assess how the four features contribute to control catalysis by performing catalytic studies under single- and limited-turnover conditions with native and chimeric NOSs of each isoform. Together, our study will provide a basis to understand how NOS isoforms perform their unique biologic functions, and may lead to new approaches for selective control.
Showing the most recent 10 out of 97 publications
