The ability of individual cells to communicate with each other is a fundamental property that allows them to coexist in a context of multicellular organisms. Nitric oxide (NO) is one of the signaling molecules used to communicate with adjacent cells. A special heterodimeric hemeprotein - soluble guanylyl cyclase (sGC) - converts this extracellular NO signal into intracellular messenger 3'-5' cyclic guanosine monophosphate. Although there are many examples of hemeproteins inhibited by interaction with NO, sGC is the only hemeproteins which is activated by NO binding. Although the function of sGC as NO receptor is well established, the processes of catalytic activation, signal coupling and enzyme deactivation are far from understood. Moreover, it is not understood how this hemeprotein which function in oxygen-rich environment selectively binds NO with high affinity, but does not bind oxygen. The proposal is based on a central hypothesis that the ligand specificity is determined by the heme pocket scaffold shaped by both sGC subunits and overall activity of the enzyme is affected by the changes of heme conformation and ligation and/or redox state induced by NO and other effector molecules. Using a combination of spectroscopic methods (UV-Vis, fluorescence, EPR, resonance Raman) we will assess the binding mechanism and catalytic effect of gaseous ligands (NO, CO, O2) to ferrous sGC and anionic ligands with different geometry to ferric sGC. To investigate the coupling mechanism between the binding of NO and cGMP synthesis we will spectroscopically monitor the processes of NO binding, transition of NO-heme complexes, protein conformational changes and will correlate this information with the dynamics of cGMP formation. The amplification ratio between NO binding and cGMP formation will be determined in studies done under single or few turnovers. To analyze the mechanism of sGC deactivation we will record the deactivation kinetics of NO-sGC complex by NO scavenger oxyhemoglobin using optical and EPR spectroscopy coupled with measurements of changes in enzyme activity. We will also investigate the changes in the red-ox state of the ligated and unligated heme during activation/deactivation cycle to test whether this is the driving mechanism of NO-dependent regulation. Intellectual Merit: The main intellectual merit of the proposal is elucidation of the novel and unique molecular mechanisms that govern the function of this enzyme, including specific ligand selection and NO induced enzyme activation. This proposal will provide incisive insight into the biophysical and biochemical specifics of NO-dependent signal transduction from the first NO binding to the final cGMP formation steps. ? ? Broader Impacts: In addition, understanding of the sGC ligand selectivity will provide new fundamental knowledge about the interaction of gaseous molecules with natural sensor and provide technical knowledge to design new nanosensors in high-sensitivity devices detecting the presence of toxic gases. Understanding of the mechanisms that govern activation of sGC may enable design for new drugs targeting the NO/cGMP- dependent signaling. The project will serve as a vehicle for training postdoctoral and graduate students. The goal of the training is to develop the student's and fellow's technical and critical thinking and reinforce their interest in science. Soluble guanylyl cyclase is a key enzyme in regulation of vascular smooth muscle relaxation, blood pressure, platelet aggregation, angiogenesis etc. In this proposal we will determine and analyze the factors crucial for different phases of sGC activity cycle. Understanding the mechanisms governing sGC function (ligand selectivity, enzyme activation or deactivation) is essential for improving existing regimens and developing new sGC-directed therapies. ? ? ?
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