Characterization of nitric oxide synthases

Fascinating research of the last decade in areas as diverse as human memory, penile erection and immune response has revealed an important transduction mechanism in which nitric oxide (NO) acts as a cellular messenger. In the vascular system. NO acts on blood vessels, especially arteries, and keeps them patent against overwhelming odds in a number of diseases such as hardening of the vessel wall (atherosclerosis), diabetic angiopathy, and lack of oxygen (ischemic syndromes). The current project aims at enhancing our understanding of NO biosynthesis. NO is synthesized from the amino acid L-arginine by a family of enzymes termed NO syntheses (NOS). Three major isoforms of NOS have been described so far: a Ca2+-dependent enzyme mainly expressed in neuronal cells, a Ca2+-dependent enzyme first identified in endothelial cells, and a Ca2+-indeperident enzyme first purified from activated macrophages. All three isoforms are homodimeric proteins that may dissociate into inactive monomers under certain conditions. NOS catalysis is a complex redox process that is not well understood. Oxidation of L-arginine occurs in two steps via formation of the intermediate NG -hydroxy-L-arginine, which undergoes immediate oxidative cleavage to NO and L-citrulline. Both reaction steps are catalyzed by a cytochrome P450-type prosthetic heme group located in the N-terminal half of the protein, designated as the oxygenase domain. The electrons required for reductive activation of O2 , which serves as a co-substrate, are provided by the nucleotide cofactor NADPH and shuttled to the heme through the flavins FAD and FMN located in the C-terminal reductase domain. Unlike other P450s, NOS requires the pterin derivative tetrahydrobiopterin (H4 biopterin) for catalytic activity. The function of H4 biopterin in NO biosynthesis is not well understood, but there is good evidence that it acts as both allosteric modulator of the protein and as redox-active participant in L-arginine oxidation. With the studies proposed here we will continue our successful research on two major topics of NOS enzymology: the function of H4biopterin in enzyme catalysis and the role of heme in the assembly of the active dimeric protein. In addition we will continue with our work on the interaction of NOS with new drugs and synthetic peptides corresponding to specific sequences of proteins suggested to interact with NOS in intact cells. To better understand the function of H4 biopterin, we will test several structural analogs of the natural cofactor for their effects on the structure and function of the different NOS isoforms. These experiments are expected to shed light on the structural features of the pterins required for the specific binding to NOS and the redox properties essential for enzyme activation. The studies on the role of heme involve biochemical and biophysical experiments with different metal porphyrins and mutated NOS proteins. It will be investigated how binding of the metal porphyrins triggers the assembly of NOS subunits and other structural changes eventually leading to enzyme activation.

Nitric oxide (NO) is a wide-spread biological signal molecule which is involved in biological processes as diverse as regulation of blood pressure, platelet aggregation, neurotransmission in the brain, penile erection, and immune defense against invading pathogens. Besides these important physiological functions, overproduction of NO may be deleterious and contribute to tissue injury in various inflammatory, infectious, and neurodegenerative diseases. Unlike other cellular messenger molecules, which act via specific interactions with ligand binding sites of target protein (enzymes, receptors, membrane transporters), nitric oxide (NO) exerts its effects through chemical reactions with cellular constituents, rendering the biological chemistry of NO important for the understanding of NO physiology and pathophysiology. In this project we studied the mechanism and biological significance of a particular chemical reaction of NO, called S-nitrosation, which, resembling the well known N-nitrosation reactions leading to the formation of carcinogenic nitrosamines, may confer potentially harmful modifications of cellular constituents containing essential sulfhydryl moieties (proteins or low-molecular mass thiols such as glutathione). In particular, we were interested in the biological conditions favoring S-nitrosation reactions and the interplay between S-nitrosation and the physiological actions of NO mediated by the most prominent target of NO, an enzyme called soluble guanylate cyclase, which converts GTP to the cyclic nucleotide cyclic GMP (cGMP) when activated by NO. The most important result we obtained refers to the potential mechanisms of cellular S-nitrosation. We identified two significant pathways of S-nitrosation, one triggered by NO autoxidation, the other triggered by co-generation of NO and superoxide anions. The autoxidation pathway takes place in cells in the presence of oxygen under conditions of sustained overproduction of NO, as occurring in inflamed tissues or upon induction of NO synthesis by bacterial infections. Using sophisticated biophysical techniques, we were able to demonstrate that nitrogen dioxide radical is the major active species which triggers S-nitrosation reactions in the course of NO autoxidation. The second pathway we have identified occurs at much lower NO concentrations but requires the additional presence of superoxide radicals, which are well known reactive species generated in inflamed tissues. According to our results, NO and superoxide combine to form the potent oxidant peroxynitrite, which oxidizes thiol anions to the corresponding thiyl radicals which rapidly react with NO to form S-nitrosothiols. So far, our studies with soluble guanylate cyclase did not provide evidence for an involvement of S-nitrosation in the physiological functions of NO, suggesting that drugs interfering with S-nitrosation could be therapeutically useful for the treatment of diseases associated with NO overproduction and nitrosative stress. The beneficial effects of several lead compounds, in particular polyphenolic scavengers of nitrogen dioxide radicals, are currently studied in our laboratory.