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

• Published in: Chemistry : a European journal 2021-01, Vol.27 (5), p.1795-1809
• Main Authors: Ali, Hafiz Saqib, Henchman, Richard H., Visser, Sam P.
• Format: Article
• Language: English
• Subjects: Arginine; Arginine - chemistry; Arginine - metabolism; Binding; Blood pressure; Catalytic Domain; Chemistry; Density functional theory; enzyme mechanism; enzyme modelling; Enzymes; Hydrogen - chemistry; Hydrogen - metabolism; Hydrogen bonds; Hydroxylation; Iron; Nitric oxide; Nitric-oxide synthase; nonheme; Nonheme Iron Proteins - metabolism; Oxidation; Oxidation-Reduction; Oxygenation; Selectivity; Substrates
• ISSN: 0947-6539; 1521-3765
• DOI: 10.1002/chem.202004019

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 ion. 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 ion pathways drive the reaction with an initial C3−H activation to a low‐energy 5σ‐pathway, while substrate positioning destabilizes the C4−H ion and sends it over the higher‐lying 5π‐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. Density functional theory calculations on the arginine dihydroxylating dioxygenase OrfP focus on the mechanism of arginine activation. We find a selective initial C3−H oxygenation pathway followed by a second oxygenation cycle on the C4−H group. A comparison with other arginine activating enzymes highlight a tight substrate binding pocket that locks the product and enables two oxygenation cycles.