文摘
Nitric oxide synthase (NOS) catalyzes the conversion of l-arginine to l-citrulline and NO in a two-step process involving the intermediate N蠅-hydroxy-l-arginine (NHA). It was shown that Cpd I is the oxygenating species for l-arginine; the hydroperoxo ferric intermediate is the reactive intermediate with NHA. Methylation of the N蠅-OH and N蠅-H of NHA significantly inhibits the conversion of NHA into NO and l-citrulline by mammalian NOS. Kinetic studies now show that N蠅-methylation of NHA has a qualitatively similar effect on H2O2-dependent catalysis by bacterial gsNOS. To elucidate the effect of methylating N蠅-hydroxy l-arginine on the properties and reactivity of the one-electron-reduced oxy-heme center of NOS, we have applied cryoreduction/annealing/EPR/ENDOR techniques. Measurements of solvent kinetic isotope effects during 160 K cryoannealing cryoreduced oxy-gsNOS/NHA confirm the hydroperoxo ferric intermediate as the catalytically active species of step two. Product analysis for cryoreduced samples with methylated NHA鈥檚, NHMA, NMOA, and NMMA, annealed to 273 K, show a correlation of yields of l-citrulline with the intensity of the g 2.26 EPR signal of the peroxo ferric species trapped at 77 K, which converts to the reactive hydroperoxo ferric state. There is also a correlation between the yield of l-citrulline in these experiments and kobs for the H2O2-dependent conversion of the substrates by gsNOS. Correspondingly, no detectable amount of cyanoornithine, formed when Cpd I is the reactive species, was found in the samples. Methylation of the NHA guanidinium N蠅-OH and N蠅-H inhibits the second NO-producing reaction by favoring protonation of the ferric-peroxo to form unreactive conformers of the ferric-hydroperoxo state. It is suggested that this is caused by modification of the distal-pocket hydrogen-bonding network of oxy gsNOS and introduction of an ordered water molecule that facilitates delivery of the proton(s) to the one-electron-reduced oxy-heme moiety. These results illustrate how variations in the properties of the substrate can modulate the reactivity of a monooxygenase.