New roles for NO in mammalian physiology are discovered daily; it is now known that NO participates in diverse functions such as blood pressure regulation, inhibition of blood clotting, immune defense, olfaction, learning and memory formation, penile erection, and digestion. NO is implicated in the pathology of diseases such as diabetes, septic shock, cancer, stroke, ischemia, and atherosclerosis. At present the enzyme soluble guanylyl cyclase (sGC) is the only confirmed cellular receptor for NO and many of the known physiological effects of NO are mediated by this enzyme. Understanding the mechanism by which NO activates its cellular target is critical given the importance of NO as a cellular signaling agent. When NO binds to the heme moiety of sGC the enzyme becomes activated: our objective is to explore the structural changes at the heme site that are associated with activation. Based on our initial spectroscopic studies, we hypothesize that the labilization of the ligand trans to NO is the trigger for activation. We propose that the activated conformation is stabilized when two conditions are met: l) neither axial ligand is bound to a metal, and 2) a porphyrin is bound in the heme site. We hypothesize that deactivation of the enzyme occurs when the proximal iron-histidine bond is reformed.
The Specific Aims of this proposal are to test these hypotheses using enzymological and spectroscopic methods. Our first specific aim is to test the hypothesis that labilization of the bond trans to NO is the trigger for activation of sGC. We will structurally characterize metalloporphyrin-substituted sGC by MCD, resonance Raman and ESR spectroscopies, to determine the actual geometry of the metalloporphyrin in these proteins and correlate this information with the activation data and theoretical models. We will further test the trans labilization hypothesis by reconstituting the protein with other metalloporphyrins having known coordination preferences. Our second specific aim is to test the hypothesis that the heme periphery is important in stabilizing the activated conformation by probing the heme site in the activated and unactivated states. We will determine the polarity of the heme binding site and measure the mobility of porphyrins bound in the heme site using fluorescence spectroscopy. Metalloporphyrin- substituted sGC will be used to identify peripheral protein-porphyrin contacts by resonance Raman spectroscopy. Our third specific aim is to test our hypothesis that deactivation of the enzyme requires reforming the proximal iron-histidine bond. We will study the kinetics and mechanism of deactivation by redox active agents by visible spectroscopy, chemical analysis and activity measurements. Our fourth specific aim is to identify new potential physiological regulators for sGC and to further characterize the inhibitor protein and deactivator molecule that we have discovered.
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