STOPPED-FLOW ANALYSIS OF CO AND NO BINDING TO INDUCIBLE NITRIC-OXIDE SYNTHASE

The oxygenase domain (amino acids 1-498) of inducible nitric oxide syn thase (iNOS(ox)) is a hemeprotein that binds L-arginine (L-Arg) and te trahydrobiopterin (H4B). During NO synthesis, the heme iron must bind and activate O-2, but it also binds self-generated NO to form an inact ive complex. To better understand how L-Arg and H4B affect heme iron f unction in iNOS(ox), we utilized stopped-flow spectroscopy to study he me reactivity with CO and NO and the properties of the resulting CO an d NO complexes. CO and NO binding to ferrous and ferric (NO only) iNOS (ox) and subsequent complex stability was studied under four condition s: in the absence of L-Arg and H4B and in the presence of either or bo th molecules. Ferric iNOS(ox) without L-Arg or H4B was dimeric and con tained low-spin heme iron, while in H4B- or L-Arg-saturated iNOS(ox), the heme iron was partially or almost completely high-spin, respective ly. In the presence of L-Arg or H4B, the rate of CO binding to ferrous iNOS(ox) was slowed considerably. indicating that these molecules res trict CO access to the heme iron. In contrast, rates of NO binding wer e minimally affected. Under all conditions, the off rates for CO and N O were very high as compared to other hemeproteins. The six-coordinate Fe-II-CO and -NO complexes that initially formed were unstable and co nverted either slowly (GO) or quickly (NO) to their respective 5-coord inate complexes. However, this transition was largely prevented by eit her L-Arg or H4B and was reversed upon air oxidation of the complex in the presence of these molecules. Thus, H4B and L-Arg both promote a c onformational change in the distal heme pocket of iNOS(ox) that can gr eatly reduce ligand access to the heme iron. The ability of H4B and L- Arg to prevent formation of a five-coordinate heme Fe-NO complex, alon g with the high off rates observed for NO, help explain why iNOS can r emain active despite forming a complex with NO during its normal catal ysis.