Stopped-flow analysis of CO and NO binding to inducible nitric oxide synthase.

The oxygenase domain (amino acids 1-498) of inducible nitric oxide synthase (iNOSox) is a hemeprotein that binds L-arginine (L-Arg) and tetrahydrobiopterin (H4B). During NO synthesis, the heme iron must bind and activate O2, but it also binds self-generated No to form an inactive complex. To better understand how L-Arg and H4B affect heme iron function in iNOSox, we utilized stopped-flow spectroscopy to study heme reactivity with CO and NO and the properties of the resulting CO and NO complexes. CO and NO binding to ferrous and ferric (NO only) iNOSox and subsequent complex stability was studied under four conditions: in the absence of L-Arg and H4B and in the presence of either or both molecules. Ferric iNOSox without L-Arg or H4B was dimeric and contained low-spin heme iron, while in H4B- or L-Arg-saturated iNOSox, the heme iron was partially or almost completely high-spin, respectively. In the presence of L-Arg or H4B, the rate of CO binding to ferrous iNOSox was slowed considerably, indicating that these molecules restrict CO access to the heme iron. In contrast, rates of NO binding were minimally affected. Under all conditions, the off rates for CO and NO were very high as compared to other hemeproteins. The six-coordinate FeII-CO and -NO complexes that initially formed were unstable and converted either slowly (CO) or quickly (NO) to their respective 5-coordinate complexes. However, this transition was largely prevented by either 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 conformational change in the distal heme pocket of iNOSox that can greatly 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, along with the high off rates observed for NO, help explain why iNOS can remain active despite forming a complex with NO during its normal catalysis.