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Kinetic and spectroscopic studies of N694C lipoxygenase: a probe of the substrate activation mechanism of a nonheme ferric enzyme.

ABSTRACT: Lipoxygenases (LOs) comprise a class of substrate activating mononuclear nonheme iron enzymes which catalyze the hydroperoxidation of unsaturated fatty acids. A commonly proposed mechanism for LO catalysis involves H-atom abstraction by an FeIII-OH- site, best described as a proton coupled electron transfer (PCET) process, followed by direct reaction of O2 with the resulting substrate radical to yield product. An alternative mechanism that has also been discussed involves the abstraction of a proton from the substrate by the FeIII-OH leading to a sigma-organoiron intermediate, where the subsequent sigma bond insertion of dioxygen into the C-Fe bond completes the reaction. H-atom abstraction is favored by a high E(o) of the FeII/FeIII couple and high pK(a) of water bound to the ferrous state, while an organoiron mechanism would be favored by a low E(o) (to keep the site oxidized) and a high pK(a) of water bound to the ferric state (to deprotonate the substrate). A first coordination sphere mutant of soybean LO (N694C) has been prepared and characterized by near-infrared circular dichroism (CD) and variable-temperature, variable-field (VTVH) magnetic circular dichroism (MCD) spectroscopies (FeII site), as well as UV/vis absorption, UV/vis CD, and electron paramagnetic resonance (EPR) spectroscopies (FeIII site). These studies suggest that N694C has a lowered E degrees of the FeII/FeIII couple and a raised pKa of water bound to the ferric site relative to wild type soybean lipoxygenase-1 (WT sLO-1) which would favor the organoiron mechanism. However, the observation in N694C of a significant deuterium isotope effect, anaerobic reduction of iron by substrate, and a substantial decrease in k(cat) (approximately 3000-fold) support H-atom abstraction as the relevant substrate-activation mechanism in sLO-1.

PROVIDER: S-EPMC2896304 | BioStudies |

REPOSITORIES: biostudies

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