Generation of reactive oxygen species and intracellular signal transduction mediated by nitric oxide synthases

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There are three types of nitric oxide synthases (NOSs) asnd their subunits are comprised of a N-terminal oxygenase domain that is linked to a C-termmal flavoprotein domain by a central calmodulin binding sequence. NOS utilizes L-arginine and N-hydroxy-L-arginine to generate NO. Under a certein condition such as the absence of substrate, electrons from NADPH are wasted to produce superoxide, H_2O_2 or H_2O, which is called as an uncoupling reaction. These reduced oxygen species (ROS) are generated at different branch points in the process, and their production ratio depends on the characteristics of the individual enzyme. It is important to elucidate the mechanisms of uncoupling in each NOS. Our present study addresses this issue by comparing uncoupled electron flux in the three NOSs and their product ratios for superoxide, H_2O_2, and water, and by examining how these are affected by certain substrate analogs. We studied ROS generation in three NOS isoforms by measuring NO, superoxide, … More and H_2O_2 production at 25℃ using oxyhemoglobin oxidation, reduction of succinylated cytochrome c, and the generation of Fe(SCN)_4^- from Fe^<2+>, respectively. NADPH consumption was also measured to determine total electron flux. In the presence of excess L-arginine and tetrahydrobiopterin, NOS1 and NOS2 did not show significant uncoupling but for NOS3 almost half of the NADPH-derived electrons went toward ROS generation during NO synthesis. In the absence of L-arginine the major ROS generated was superoxide for NOS1 and NOS3 but was H_2O_2 for NOS2. Adding pseudo-substrates that increased or decreased electron flux through the NOS heme did not greatly alter ROS product ratios. Their pattern of ROS formation distinguish NOSs from cytochrome P450's which generate mainly H_2O_2 and water during uncoupled NADPH consumption. The different ROS product ratio of NOS1 and NOS2 is surprising, and suggests how variation at discreet steps in the oxygen activation pathway can impact NOS-related inflammation processes. Less