Enzymatic and cryoreduction EPR studies of the hydroxylation of methylated N(ω)-hydroxy-L-arginine analogues by nitric oxide synthase from Geobacillus stearothermophilus.

Nitric oxide synthase (NOS) catalyzes the conversion of l-arginine to l-citrulline and NO in a two-step process involving the intermediate Nω-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 Nω-OH and Nω-H of NHA significantly inhibits the conversion of NHA into NO and l-citrulline by mammalian NOS. Kinetic studies now show that Nω-methylation of NHA has a qualitatively similar effect on H2O2-dependent catalysis by bacterial gsNOS. To elucidate the effect of methylating Nω-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 wit...