Spectroscopic and Functional Characterization of a Ligand Coordination Mutant of Soybean Lipoxygenase-1: First Coordination Sphere Analogue of Human 15-Lipoxygenase

Lipoxygenases are an important class of non-heme iron enzymes which catalyze the hydroperoxidation of unsaturated fatty acids. This study utilizes a combination of electron paramagnetic resonance (EPR), magnetic circular dichroism (MCD), and variable-temperature, variable-field (VTVH) MCD to probe the ground state and excited states of the active site of an iron coordination mutant of soybean lipoxygenase-1 (SLO-1), N694H, and relate the structural modifications with kinetic changes. The sixth coordination ligand for SLO-1, Asn694, is proposed to be a primary factor in the coordination flexibility of the site and is reflected in both its ferrous MCD and ferric EPR spectroscopic properties. In the present study, we compare the spectroscopic properties of the N694H SLO-1 mutant with those of wild-type (WT) SLO-1 and WT human 15-lipoxygenase (15-HLO) and determine that its properties are intermediate between the WT enzymes. WT 15-HLO has a His substitution in the Asn694 equivalent position. The low-temperatu...