Molecular function of nitric oxide synthase (NOS): Direct electrochemical investigation

Nitric oxide (NO) is one of the smallest molecules produced in vivo, yet it carries the most profound effects on regulatory mechanisms of the cardiovascular system and other critical functions of the body. NO is a key signaling agent in regulation of smooth muscle relaxation and vasodilation and in inhibition of platelet adhesion and aggregation. This molecule is also involved in neurotransmission and it is vital for the body's immune response. NO is produced by a class of enzymes called nitric oxide synthases (NOSs). NOS enzymes belong to a super-family of complex flavo-heme enzymes, which carry out the five-electron oxidation of the substrate L-arginine to the product NO, and the by-product, L-citrulline. The reaction takes place on a Fe-heme center and involves more than one component to achieve the desired reaction. Electron transfer events are of course critical to this enzymatic reaction and are at least partially rate-determining. This work explores electron transport and direct electrochemical properties of the NOS heme enzymes in bilayered thin films on solid electrodes. Our study addresses the factors that modulate the thermodynamics and the kinetics of electron transfers to and from the heme in the oxygenase center. We also use our direct electrochemical approach to activate molecular oxygen and to carry out the full NOS turnover on NOS-modified electrodes. Our direct electrochemistry approach, combined with site-directed mutagenesis, is used to develop molecular-level understanding of key elemental steps of the NOS reaction.