The binding sites of nitric oxide synthases

Nitric oxide synthases (NOSs) are a family of three related gene products that catalyze the biosynthesis of nitric oxide, an important biosignaling molecule. All NOSs require five cofactors or prosthetic groups for activity. Herein, we have studied binding and regulation of NOS activity by two of them, calmodulin (CaM) and tetrahydrobiopterin (BH{dollar}sb4).{dollar} Identification of similarities and differences between NOS isoforms were a focus of this study.; Reversible activation by Ca{dollar}sp{lcub}2+{rcub}{dollar}-dependent CaM binding is a feature shared by constitutive NOS isoforms (cNOSs), but not the inducible isoform (iNOS). The molecular basis for the activation process and its regulation was investigated. Characterization of Ca{dollar}sp{lcub}2+{rcub}{dollar}-dependent CaM binding and activation of cNOSs suggested that cNOS activation is a sequential process, starting with the anchoring of CaM's C-lobe and accomplishing by the binding of CaM's N-lobe. Occupation of the CaM binding site is necessary but insufficient to elicit activation. Furthermore, a peptide insertion in cNOS FMN domain was identified as an autoinhibitory control element which apparently functions as a molecular switch. Binding of CaM to cNOSs was shown to elicit a conformational change which displaces the autoinhibitory module, thus activating cNOSs by disinhibition.; Both binding of BH{dollar}sb4{dollar} to its binding site and location of the site on NOSs were examined. Binding of BH{dollar}sb4{dollar} was found to be affected by NOS substrate L-arginine (Arg). Conversely, binding of the Arg analog (NNA) to NOSs was shown to be dependent on the presence of BH{dollar}sb4,{dollar} revealing that one function of BH{dollar}sb4{dollar} in NOSs is to allow for substrate binding by allosteric regulation. In addition, binding sites for both BH{dollar}sb4{dollar} and Arg were formally confirmed to reside in NOS's N-terminal oxygenase domain, a domain which was also found to contain the dimerization site and to be catalytically competent when incubated with suitable electron donor. Arg and BH{dollar}sb4{dollar} binding sites were further localized to nNOS{dollar}sb{lcub}558-721{rcub}{dollar} and nNOS{dollar}sb{lcub}220-506{rcub},{dollar} respectively. Structural features of BH{dollar}sb4{dollar} analogs necessary to support substrate binding and NOS activity were also examined and found to differ subtly between NOS isoforms. These studies also led to the conclusion that BH{dollar}sb4{dollar} is not merely an allosteric regulator of NOS activity but may also play a redox-active role in NOS catalysis.