Three nitric oxide synthase enzymes (NOS) evolved to function in human health and disease. We wish to define the mechanisms and the protein structural features that regulate NOS catalysis. The NOS flavoprotein domain transfers NADPH electrons to a heme in the oxygenase domain and this enables NO synthesis. Catalytic regulation is complex because NO binds to the NOS heme before it exits the enzyme. We developed a global kinetic model that incorporates these and other facets of catalysis. The global model posits that the unique catalytic profiles of the three NOS are due to differences in three kinetic parameters: Ferric heme reduction (kr), ferric heme-NO dissociation (kd), and oxidation of the ferrous heme-NO complex (kox). The mechanisms and structural basis for controlling NOS electron transfer and for setting the kr, kd, and kox values are still unclear.
Our Aims propose biochemical, kinetic, molecular biological, and biophysical studies to tackle these issues, in order to provide a molecular-level understanding of NOS control mechanisms.
Aim 1. What regulates electron transfer and heme reduction (kr) in NOS? Electron transfer by the FMN module is a central feature of NOS catalysis. We will investigate how partner subdomains, FMN redox status, &calmodulin regulate the conformational equilibrium of the FMN module and its interactions with electron acceptors, how inter-domain surface charge interactions regulate FMN electron transfer and NOS catalysis, and how a specific FMN-protein interaction and the composition of two connecting hinge elements govern FMN module electron transfer in NOS.
Aim 2. What regulates reaction of the ferrous heme-NO complex with O2 (kox)? The kox reaction is key because it determines NOS catalytic behavior and generates an N-oxide product that is distinct from NO. We will investigate how kox may be regulated by the NOS heme midpoint potential, protein structural features that control O2 access to the heme, and interactions between the FMN and NOSoxy subdomains, and will study the kox mechanism and regulation in several related heme-thiolate enzymes.
Aim 3. How do NOS enzymes regulate their NO release (kd)? Factors that control ferric heme-NO dissociation (kd) are particularly important for NOS because its newly-generated NO molecules coordinate to the ferric heme before leaving the enzyme. We will investigate how the NO kd is controlled by NOS dimeric structure, substrate binding &structure, and the size of the heme pocket opening.
Aim 4. Generate NOS with novel catalytic behaviors through protein engineering. Within the context of Aims 1-3 we will create NOS variants that possess non- native combinations of kr, kox and kd. These will test the global model and our understanding of kinetic control mechanisms in NOS, will reveal if NOS enzyme function can change when the kinetic parameters exceed their natural ranges, and may generate useful super-NOS variants for NO delivery therapy. Relevance: By clarifying how nitric oxide production is regulated at the enzyme level, our work may help to develop treatments for human diseases that involve making too much or too little nitric oxide.

Public Health Relevance

Three nitric oxide synthase enzymes function broadly in human health and disease. We wish to determine how the protein components of these enzymes regulate their nitric oxide production. By clarifying how nitric oxide production is regulated at the enzyme level, our work may help to develop treatments for human diseases that involve making too much or too little nitric oxide.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM051491-16
Application #
8112754
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Anderson, Vernon
Project Start
1994-08-01
Project End
2013-07-31
Budget Start
2011-08-01
Budget End
2013-07-31
Support Year
16
Fiscal Year
2011
Total Cost
$346,221
Indirect Cost
Name
Cleveland Clinic Lerner
Department
Other Basic Sciences
Type
Schools of Medicine
DUNS #
135781701
City
Cleveland
State
OH
Country
United States
Zip Code
44195
Dai, Yue; Haque, Mohammad Mahfuzul; Stuehr, Dennis J (2017) Restricting the conformational freedom of the neuronal nitric-oxide synthase flavoprotein domain reveals impact on electron transfer and catalysis. J Biol Chem 292:6753-6764
AlTawallbeh, Ghaith; Haque, Mohammad M; Streletzky, Kiril A et al. (2017) Endothelial nitric oxide synthase oxygenase on lipid nanodiscs: A nano-assembly reflecting native-like function of eNOS. Biochem Biophys Res Commun 493:1438-1442
Haque, Mohammad Mahfuzul; Ray, Sougata Sinha; Stuehr, Dennis J (2016) Phosphorylation Controls Endothelial Nitric-oxide Synthase by Regulating Its Conformational Dynamics. J Biol Chem 291:23047-23057
Ramasamy, Somasundaram; Haque, Mohammad Mahfuzul; Gangoda, Mahinda et al. (2016) Tetrahydrobiopterin redox cycling in nitric oxide synthase: evidence supports a through-heme electron delivery. FEBS J 283:4491-4501
Rwere, Freeborn; Xia, Chuanwu; Im, Sangchoul et al. (2016) Mutants of Cytochrome P450 Reductase Lacking Either Gly-141 or Gly-143 Destabilize Its FMN Semiquinone. J Biol Chem 291:14639-61
Sarkar, Anindya; Dai, Yue; Haque, Mohammad Mahfuzul et al. (2015) Heat Shock Protein 90 Associates with the Per-Arnt-Sim Domain of Heme-free Soluble Guanylate Cyclase: IMplications for Enzyme Maturation. J Biol Chem 290:21615-28
Hannibal, Luciana; Page, Richard C; Haque, Mohammad Mahfuzul et al. (2015) Dissecting structural and electronic effects in inducible nitric oxide synthase. Biochem J 467:153-65
He, Yufan; Haque, Mohammad Mahfuzul; Stuehr, Dennis J et al. (2015) Single-molecule spectroscopy reveals how calmodulin activates NO synthase by controlling its conformational fluctuation dynamics. Proc Natl Acad Sci U S A 112:11835-40
Ghosh, Arnab; Stasch, Johannes-Peter; Papapetropoulos, Andreas et al. (2014) Nitric oxide and heat shock protein 90 activate soluble guanylate cyclase by driving rapid change in its subunit interactions and heme content. J Biol Chem 289:15259-71
Haque, Mohammad M; Bayachou, Mekki; Tejero, Jesus et al. (2014) Distinct conformational behaviors of four mammalian dual-flavin reductases (cytochrome P450 reductase, methionine synthase reductase, neuronal nitric oxide synthase, endothelial nitric oxide synthase) determine their unique catalytic profiles. FEBS J 281:5325-40

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