Kinetics of CO binding to the haem domain of murine inducible nitric oxidesynthase: differential effects of haem domain ligands

The binding of CO to the murine inducible nitric oxide synthase (iNOS) oxyg enase domain has been studied by laser flash photolysis. The effect of the (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4) cofactor L-arginine and several T ype I L-arginine analogues/ligands on the rates of CO rebinding has been ev aluated. The presence of BH4 in the iNOS active site has little effect on t he rebinding of protein-caged haem CO pairs (geminate recombination), but d ecreases the bimolecular association rates 2-fold. Addition Of L-arginine t o the BH4-bound complex completely abolishes geminate recombination and res ults in a further SO-fold decrease in the overall rate of bimolecular assoc iation. Three of the Type I ligands, S-ethylisothiourea, L-canavanine and 2 ,5-lutidine, displaced the CO from the haem iron upon addition to the iNOS oxygenase domain. The Type I ligands significantly decreased the rate of bi molecular binding of CO to the haem iron after photolysis. Most of these li gands also completely abolished geminate recombination. These results are c onsistent with a relatively open distal pocket that allows CO to bind unhin dered in the active site of murine iNOS in the absence of L-arginine or BH4 . In the presence of BH4 and L-arginine, however, the enzyme adopts a more closed structure that can greatly reduce ligand access to the haem iron. Th ese observations are discussed in terms of the known structure of iNOS haem domain and solution studies of ligand binding in iNOS and neuronal NOS iso enzymes.