CHARACTERIZATION OF THE INACTIVATION OF NITRIC-OXIDE SYNTHASE BY N-G-METHYL-L-ARGININE - EVIDENCE FOR HEME LOSS

The nitric oxide synthases (NOS) are a unique family of P450-type hemo proteins that catalyze the formation of .NO and citrulline from L-argi nine, oxygen, and NADPH. NG-Methyl-L-arginine (L-NMA) has been shown t o function as a slow, partially uncoupled alternate substrate and mech anism-based inhibitor of the inducible NOS [Olken, N. M., and Marletta , M. A. (1993) Biochemistry 32, 9677-9685]. In this report, the inacti vation of NOS by L-NMA has been investigated in detail. Inactivation f ails to occur under an argon atmosphere, establishing turnover depende nce. The partition ratio, defined as the number of molecules of citrul line formed per NOS monomer inactivated, is 108 +/- 3. By utilizing N- G-methyl-L-[2,3-H-3(2)]arginine and N-G-[C-14]methyl-L-arginine, the s toichiometry of radiolabeling is 0.11 +/- 0.01 equiv of tritium and 0. 41 +/- 0.10 equiv of carbon-14 per inactivated NOS monomer. Dialysis u nder native conditions does not change this stoichiometry. However, di alysis of NOS following denaturation decreases the stoichiometry of ra diolabeling to 0.08 +/- 0.04 equiv of tritium and 0.12 +/- 0.04 equiv of carbon-14 per inactivated NOS monomer. Absolute and CO-reduced diff erence spectroscopy indicates that inactivation of L-NMA is accompanie d by a substantial loss of the heme chromophore, which is not prevente d by catalase. HPLC analysis of NOS heme following inactivation with L -NMA indicates substantial loss of heme. These findings suggest that m ultiple mechanisms may contribute to the loss of NOS activity by L-NMA , including heme loss and possibly protein and cofactor modification.