Distal Val346Ile mutation in inducible NO synthase promotes substrate-dependent NO confinement.

The function of inducible NO synthase (WT iNOS) depends on the release of NO from the ferric hemebefore the enzyme is reduced. Key parameters controlling ligand dynamics include the distal and proximal hemepocket amino acids, as well as the inner solvent molecules. In this work, we tested how a point mutation in the distal hemeside of WT iNOS affected the geminaterebinding of NO by ultrafast kinetics and molecular dynamics simulations. The mutation sequestered much of the photodis- sociated NO close to the hemecompared to WT iNOS, with a main picosecond phase accounting for 78% of the rebinding to the arginine-bound Val346Ile protein. Consequently, the probability of NO release from Val346Ile decreased as compared to that from WT iNOS, provided the substrate binding site is filled. These data are rationalized by a steric effectof the Ile methyl group inducing events mediated by the substrate, transmitted via the propionates to the NO and the protein. This model is consistent with the role of the H-bonding network involving the heme, the substrate, and the BH4 cofactor in controlling NO release, with a key role of the hemepropionates (Gautier et al. (2006) Nitric Oxide 15, 312). These data support the effect of Val346Ile mutation in decreasing NO release and slowing down NO synthesis compared to WT iNOS determined by single turnover catalysis (Wang et al. (2004) J. Biol. Chem. 279, 19018). ‡