The overall goal of this proposal is to provide a molecular understanding of the functional role of tetrahydrobiopterin (BH4) and the overall reaction mechanism of endothelial-type nitric oxide synthase (eNOS). The mechanistic hypothesis to be tested is based on a 3/2 coupling model between the reductase and the P450 oxidase mediated by calmodulin, CaM/Ca +2.To test the first hypothesis that efficient redox cycling between BH4 and its radical intermediate warrants a NO synthase rather than a superoxide synthase activity, we will obtain complete kinetic data of different biopterin redox species including its radical intermediate during single and multiple turnover reaction of eNOS. The kinetics of the biopterin radical will be correlated with redox change of other redox centers and the substrate disappearance and product formation to resolve the full catalytic mechanism of the oxygenase domain. To address the protein and heme effect on the biopterin redox behavior site-specific mutation of critical residues involved in BH4 binding and heme reactivity will be generated. Several BH4 analogs will be utilized in the kinetic experiments described in Aim 1. The structure of the biopterin radical will be characterized by different spectroscopic methods and DFT calculations. To test the last hypotheses that redox state of the flavins and the binding of CaM have deciding effect on the overall turnover rate and coupling of the reductase and oxygenase activities, we will look into the oxidative and reductive reaction mechanism of the reductase domain in the presence and absence of CaM. We wish to locate the rate-limiting step(s), the half-reaction that couples to heme reduction, defining the internal electron transfer and conformational gating. Domain communication will be assessed by manipulating the autoinhibitory sequence and to evaluate the effect of CaM on electron transfer.
The first Aim i s to address the reaction mechanism of the oxygenase domain and the function of BH4 as the electron donor for the two steps of oxygenase reaction, and in steering NO formation instead of nitroxyl (HNO) or superoxide (O2-) production.
The second aim will provide critical information regarding the structure/function relationship between the BH4 and heme and the factors that regulate BH4 redox behaviors. The last Aim will provide understanding of the role of reductase and CaM in regulating the redox coupling with the oxygenase domain thus lead to an elucidation of the reaction of the reaction mechanism of the whole eNOS.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM056818-08
Application #
7152901
Study Section
Special Emphasis Panel (ZRG1-SSS-B (01))
Program Officer
Ikeda, Richard A
Project Start
1999-08-01
Project End
2008-11-30
Budget Start
2006-12-01
Budget End
2008-11-30
Support Year
8
Fiscal Year
2007
Total Cost
$238,352
Indirect Cost
Name
University of Texas Health Science Center Houston
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
800771594
City
Houston
State
TX
Country
United States
Zip Code
77225
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Berka, Vladimir; Wu, Gang; Yeh, Hui-Chun et al. (2004) Three different oxygen-induced radical species in endothelial nitric-oxide synthase oxygenase domain under regulation by L-arginine and tetrahydrobiopterin. J Biol Chem 279:32243-51
Du, Mei; Yeh, Hui-Chun; Berka, Vladimir et al. (2003) Redox properties of human endothelial nitric-oxide synthase oxygenase and reductase domains purified from yeast expression system. J Biol Chem 278:6002-11

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