Nitric oxide (NO) participates both in the normal cardiac physiology and various cardiac pathological events including myocardial ischemia and reperfusion injury. Dynamic expression and activation of specific NOS isozyme occurs at different stages of the disease processes. Whether NO is cardioprotective or cardiodestructive remains controversial due to the complexity of the chemical reactions catalyzed by NOS. Changing of the choreography of the substrate supply and cofactor binding could transform NO synthase to catalyst for the reactive oxygen species (ROS) or reactive nitrogen species (RNS) that are important intermediates for cardiac pathophysiology. Our recent studies disclosed very different radical intermediate profile and regulation mechanism in eNOS and nNOS catalysis. The central hypothesis of this proposal is that understanding the interplay of the various regulatory molecules and the dynamic changes of the ROS, RNS and other radical intermediates during coupled and uncoupled NOS catalysis are crucial to elucidation of the etiology of myocardial infarction and ischemia- reperfusion injury. Furthermore, previous studies using whole tissue, cells, or purified enzyme under steady-state condition with spin-trapping are insufficient to obtain direct structural and kinetic information and require other innovative approach. We plan to elucidate the mechanism of radical intermediates dynamics in three NOS isozymes:
In Aim 1, we wish to test the hypothesis that different radical intermediates are formed in the nNOSox, eNOSox and iNOSox. Innovative rapid-freeze quench (RFQ) EPR kinetic measurements and other pulsed EPR methods will be used to characterize new radical intermediates as well as their kinetics.
In Aim 2, we will test the hypothesis that thiol is required in preventing BH4 oxidation in all NOS isoforms but is also necessary for keeping structural integrity of the nNOS and iNOS. Similar RFQ EPR kinetic measurements will be conducted in the presence and absence of thiol. Site-specific mutants will be used to assess the role of the key cysteines.
In Aim 3, we plan to test whether the reductase domain is the main source of radicals in iNOS but not eNOS or nNOS. Purified full length NOS and NOSred of three isoforms will be evaluated for oxygen-induced radical intermediates using CaM/Ca+2 or disruption of heme coordination to dissect the radical contribution from the NOSox and NOSred. Both cardiomyocytes and macrophage-like cells will be our models for ischemia/reperfusion to assess the regulatory roles of thiol, oxygen, substrate, cofactor and inhibitors on the radical intermediate profile in the last aim. These approaches will provide the most basic knowledge on the mechanism under coupled and uncoupled conditions of each NOS isoforms and can be useful in developing therapeutic regimens for treating reperfusion injury.

Public Health Relevance

This project focuses on characterizing the structure and temporal dependence of the radical intermediates, including ROS and RNS, induced by oxygen in all three nitric oxide synthase isozymes. The regulation of these radical intermediates by substrate, cofactors and thiol also are studied, both in vitro and ex vivo, in order to elucidate the underlying disease mechanism of myocardial ischemia and reperfusion injury.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Macromolecular Structure and Function E Study Section (MSFE)
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Mcdonald, Cheryl
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University of Texas Health Science Center Houston
Internal Medicine/Medicine
Schools of Medicine
United States
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Wu, Gang; Liu, Wen; Berka, Vladimir et al. (2017) Gaseous ligand selectivity of the H-NOX sensor protein from Shewanella oneidensis and comparison to those of other bacterial H-NOXs and soluble guanylyl cyclase. Biochimie 140:82-92
Krzyaniak, Matthew D; Cruce, Alex A; Vennam, Preethi et al. (2016) The tetrahydrobiopterin radical interacting with high- and low-spin heme in neuronal nitric oxide synthase - A new indicator of the extent of NOS coupling. Free Radic Biol Med 101:367-377
Wu, Gang; Liu, Wen; Berka, Vladimir et al. (2015) H-NOX from Clostridium botulinum, like H-NOX from Thermoanaerobacter tengcongensis, Binds Oxygen but with a Less Stable Oxyferrous Heme Intermediate. Biochemistry 54:7098-109
Cruce, Alex A; Lockart, Molly; Bowman, Michael K (2015) Pulsed EPR in the Study of Drug Binding in Cytochrome P450 and NOS. Methods Enzymol 563:311-40
Samuel, Errol L G; Marcano, Daniela C; Berka, Vladimir et al. (2015) Highly efficient conversion of superoxide to oxygen using hydrophilic carbon clusters. Proc Natl Acad Sci U S A 112:2343-8
da Silva, Giordano F Z; Goblirsch, Brandon R; Tsai, Ah-Lim et al. (2015) Cation-Specific Conformations in a Dual-Function Ion-Pumping Microbial Rhodopsin. Biochemistry 54:3950-9
Mollan, Todd L; Jia, Yiping; Banerjee, Sambuddha et al. (2014) Redox properties of human hemoglobin in complex with fractionated dimeric and polymeric human haptoglobin. Free Radic Biol Med 69:265-77
Berka, VladimĂ­r; Liu, Wen; Wu, Gang et al. (2014) Comparison of oxygen-induced radical intermediates in iNOS oxygenase domain with those from nNOS and eNOS. J Inorg Biochem 139:93-105
Zhang, Yujin; Berka, Vladimir; Song, Anren et al. (2014) Elevated sphingosine-1-phosphate promotes sickling and sickle cell disease progression. J Clin Invest 124:2750-61
Maryasov, Alexander G; Bowman, Michael K (2013) Bloch equations for anisotropic paramagnetic centers with spin of 1/2. J Magn Reson 233:80-6

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