Analysis of Electron Flow in Nitric Oxide Synthase

Nitric oxide synthase (NOS) transfers electrons supplied with NADPH to oxygen on heme of cytochromeP450-type and oxidizes -arginine to produce NO during enzyme turnover. But，如果activated oxygen cannot react with L-arginine，the oxygen is released from NOS as reactive oxygen species (ROS). This phenomenon which is calleduncoupling reaction,was clarified in detail in each NOS isoform and dependency upon cofactor and substrates wasdetermined. Generations of NO, superoxide (o_2 ^-)，and h_2_2 were measured at 25℃with the metHb formation from HbOthe reduction of succinyl cytochrome c, and the generation of Fe (SCN)_4 from Fe^<2+>，respectively. NADPH consumption was also measured to determine total electron flux. Under bh_4arginine-saturated condition, no - i and no - ii did not show significant uncoupling. However，in NO - iii almost a half of electrons remained unconsumed during NO synthesis. in所有NOSs，the depletion of substrate arginine led to uncoupling reactionwhose ic__ <50> corresponded to the arginine affinity to each NOS isoform. Under uncoupling condition，major ROS generated from NOS was superoxide in NOS- i and NOS- iiiwhereas h_2_2 for NOS-II.For NOS- i and NOS- ii，the change in electron transfer rate by substrates or cofactors did not alter the . o_2 ^-/ h_2_2ratio, whereas, for no - iii，the increase in electron transfer rate enhanced . o_2 - generation. These differences of three NOSisoforms may have an impact on its role in the pathogenesis of nos -相关的inflammation processes。

Nitric oxide synthase (NOS) transfers electrons supplied with NADPH to oxygen on heme of cytochrome P450-type and oxidizes L-arginine to produce NO during enzyme turnover. But, if the activated oxygen cannot react with L-arginine, the oxygen is released from NOS as reactive oxygen species (ROS). This phenomenon which is called uncoupling reaction, was clarified in detail in each NOS isoform and dependency upon cofactor and substrates was determined. Generations of NO, superoxide (・O_2^-), and H_2O_2 were measured at 25℃ with the metHb formation from HbO_2, the reduction of succinyl cytochrome c, and the generation of Fe (SCN)_4 from Fe^<2+>, respectively. NADPH consumption was also measured to determine total electron flux. Under BH_4^- and arginine-saturated condition, NOS-I and NOS-II did not show significant uncoupling. However, in NOS-III almost a half of electrons remained unconsumed during NO synthesis. In all NOSs, the depletion of substrate, arginine led to uncoupling reaction, whose IC_<50> corresponded to the arginine affinity to each NOS isoform. Under uncoupling condition, major ROS generated from NOS was superoxide in NOS-I and NOS-III, whereas it was H_2O_2 for NOS-II.For NOS-I and NOS-II, the change in electron transfer rate by substrates or cofactors did not alter the ・O_2^-/H_2O_2 ratio, whereas, for NOS-III, the increase in electron transfer rate enhanced ・O_2^- generation. These differences of three NOS isoforms may have an impact on its role in the pathogenesis of NOS-related inflammation processes.