Mechanistic Studies of Inactivation of Inducible Nitric Oxide Synthase by Amidines

Nitric oxide synthase (NOS) catalyzes the conversion of l-arginine to l-citrulline and nitric oxide. <i>N</i>⁵-(1-Iminoethyl)-l-ornithine (l-NIO), an amidine-containing molecule, is a natural product known to be an inactivator of inducible NOS (iNOS). Because of the presence of the amidine methyl group in place of the guanidine amino group of substrate l-arginine, the active site heme peroxy intermediate sometimes cannot be protonated, thereby preventing its conversion to the heme oxo intermediate; instead, a heme oxygenase-type mechanism occurs, leading to conversion of the heme to biliverdin. This might be a new and general inactivation mechanism for heme-containing enzymes. In the studies described here, we attempted to provide support for amidines as substrates and inactivators of iNOS by the design and synthesis of amidine analogues of l-NIO having groups other than the amidine methyl group. No nitric oxide- or enzyme-catalyzed products could be detected by incubation of these amidines with iNOS. Although none of the l-NIO analogues acted as substrates, they all inhibited iNOS; increased inhibitory potency correlated with decreased substituent size. Computer modeling and molecular dynamics simulations were run on <b>10</b> and <b>11</b> to rationalize why these compounds do not act as substrates. Unlike the methyl amidine (l-NIO), the other alkyl groups block binding of O₂ at the heme iron. Compounds <b>8</b>, <b>9</b>, and <b>11</b> were inactivators; however, no heme was lost, and no biliverdin was formed. No kinetic isotope effect on inactivation was observed with perdeuterated ethyl <b>8</b>. A small amount of dimer disruption occurred with these inactivators, although the amount would not account for complete enzyme inactivation. The l-NIO analogues inactivate iNOS by a yet unknown mechanism; however, it is different from that of l-NIO, and the inactivation mechanism previously reported for l-NIO appears to be unique to methyl amidines