Redox properties of heme-oxygenase in NO synthases

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DESCRIPTION (provided by applicant): Nitric oxide (NO) is undeniably one of the most important biological signaling species discovered in the recent two decades. Age-old as it seems, nitric oxide only recently emerged as a key player involved in control mechanisms of cardiovascular activities, in neurotransmission, and as potent weapon in antibacterial and antiviral action of macrophages. NO is synthesized in vivo from the amino acid L-arginine by a class of enzymes known as Nitric Oxide Synthases (NOSs) requiring a P450-heme, a biopterin cofactor and molecular oxygen in its oxygenase domain. Although the biochemistry of these enzymes has been the focus of vigorous investigations in the last decade, a lot is yet to be learned about their molecular functioning, and especially their post-translational regulation; understanding the details of these processes may open avenues for potential NOS targeted therapies. In this regard, we propose a direct-electrochemical study to investigate mechanisms of electron transfer to, and oxygen activation by nitric oxide synthases. We aim to develop fast protocols to study the effect of the cofactor tetrahydrobiopterin on the electronic properties of the heme as well as on oxygen activation in the NOS catalysis. Similarly, we want to use direct-electrochemistry to measure the effect of substrate arginine and NOS-inhibitors, especially endogenous methylarginines, on redox properties of the heme-oxygenase in NOS. To this end, we use immobilized NOS-oxygenase domain (NOSoxy) in thin films on electrodes to perform fast and direct electrochemistry (i.e. without mediators). We synergistically use computational methodologies to complement and guide our experimental endeavors. Specific aims of our proposed study are: 1) Measure thermodynamic redox potentials and kinetics of charge transfer to iron-heme in NOSoxys by direct electrochemistry and quantify the effects of binding of substrate L-arginine, cofactor biopterin, and NOS-inhibitors (such as endogenous methylarginines). 2) Compare direct electrochemical behavior of different isoforms (i.e. neuronal NOS: nNOS, inducible NOS: iNOS, etc.), as well as wild-type NOSoxys versus mutants, and correlate experimental results on oxygen activation and catalysis to computational findings.