Kinetic Study of Ligand Binding and Conformational Changes in Inducible Nitric Oxide Synthase

Nitric oxide synthases (NOSs) are heme enzymes that generate highly reactive nitric oxide from l-arginine (l-Arg) in a complex mechanism that is still only partially understood. We have studied carbon monoxide (CO) binding to the oxygenase domain of murine inducible NOS (iNOS) by using flash photolysis. The P420 and P450 forms of the enzyme, assigned to a protonated and unprotonated proximal cysteine, through which the heme is anchored to the protein, show markedly different CO rebinding properties. The data suggest that P420 has a widely open distal pocket that admits water. CO rebinding to the P450 form strongly depends on the presence of the substrate l-Arg, the intermediate N^ω-hydroxy-l-arginine, and the cofactor tetrahydrobiopterin. After adding these small molecules to the enzyme solution, the CO kinetics change slowly over the hours. This process can be described as a relaxation from a fast rebinding, metastable species to a slowly rebinding, thermodynamically stable species, which we associate with the enzymatically active form. Our results allow us to determine kinetic parameters of l-Arg binding to the ferrous deoxy iNOS protein for the first time and also provide clues regarding the nature of structural differences between the two interconverting species.