Lipoxygenase reaction mechanism: demonstration that hydrogen abstraction from substrate precedes dioxygen binding during catalytic turnover.

Molecular oxygen is generally unreactive toward covalent bonds, due to spin conservation rules; a major role for oxygen-utilizing enzymes is, therefore, to activate dioxygen through a change in electronic configuration. In an effort to understand how lipoxygenase catalyzes lipid hydroperoxidation under conditions of catalytic turnover, kinetic deuterium isotope effects have been measured as a function of oxygen concentration. The properties of oxygen binding to lipoxygenase have also been pursued. The results presented herein show that, under steady state conditions, atmospheric oxygen enters the reaction pathway only after abstraction of hydrogen from substrate. Furthermore, it has not been possible to detect any form of lipoxygenase capable of binding molecular oxygen in the absence of activated substrate. We propose that molecular oxygen is not productively bound by lipoxygenase but rather interacts directly with the substrate radical lipoxygenase to form the hydroperoxyl radical of linoleate. A mechanism involving substrate activation, instead of the more familiar oxygen activation pathway, is a unique mechanism for a metallo-oxygenase.