L-thiocitrulline: A stereospecific, heme-binding inhibitor of nitric- oxide synthases

Nitric-oxide synthase (NOS) catalyzes the oxidation of L-arginine to citrulline and nitric oxide (NO). The enzyme is inhibited by a variety of N(ω)-monosubstituted L-arginine analogs, and some of these compounds are useful in reversing pathologies associated with the overproduction of NO (e.g. the hypotension of septic shock). We report here that L-thiocitrulline (γ-thioureido-L-norvaline) is a potent, stereospecific inhibitor of the constitutive brain and endothelial isoforms of NOS as well as the isoform induced in vascular smooth muscle cells by lipopolysaccharide and interferon- γ. Steady state kinetic studies show L-thiocitrulline inhibition is competitive with L-arginine (K(i) ~ 4-20% of K(m)/(Arg)), indicating that initial binding is as a substrate/product analog. In contrast to L-arginine and N(ω)-methyl-L-arginine, the prototypic NOS inhibitor, L-thiocitrulline binding elicits a 'Type II' difference spectrum, indicating a high spin to low spin transition of the iron in the heme cofactor. This finding suggests that L-thiocitrulline is contributing the sixth ligand to heme iron, probably through the thioureido sulfur. Such interaction with heme iron neither stimulates nor inhibits the direct flavin-mediated cytochrome c reduction activity of the enzyme, but it does inhibit heme-dependent superoxide formation. In vivo, L-thiocitrulline is a potent pressor agent in both normal and endotoxemic rats, the latter finding suggesting utility in treating the hypotension of septic shock.