What Determines the Selectivity of Arginine Dihydroxylation by the Nonheme Iron Enzyme OrfP?

Abstract

The nonheme iron enzyme OrfP reacts with l-Arg selectively to form the 3R,4R-dihydroxyarginine product, which in mammals can inhibit the nitric oxide synthase enzymes involved in blood pressure control. To understand the mechanisms of dioxygen activation of l-Arg by OrfP and how it enables two sequential oxidation cycles on the same substrate, we performed a density functional theory study on a large active site cluster model. We show that substrate binding and positioning in the active site guides a highly selective reaction through C³ -H hydrogen atom abstraction. This happens despite the fact that the C³ -H and C⁴ -H bond strengths of l-Arg are very similar. Electronic differences in the two hydrogen atom abstraction pathways drive the reaction with an initial C³ -H activation to a low-energy ⁵ σ-pathway, while substrate positioning destabilizes the C⁴ -H abstraction and sends it over the higher-lying ⁵ π-pathway. We show that substrate and monohydroxylated products are strongly bound in the substrate binding pocket and hence product release is difficult and consequently its lifetime will be long enough to trigger a second oxygenation cycle.