Kinetic investigation of the rate-limiting step of manganese- and iron-lipoxygenases

Lipoxygenases (LOX) oxidize polyunsaturated fatty acids to hydroperoxides, which are generated by proton coupled electron transfer to the metal center with FeIIIOH− or MnIIIOH−. Hydrogen abstraction by FeIIIOH− of soybean LOX-1 (sLOX-1) is associated with a large deuterium kinetic isotope effect (D-KIE). Our goal was to compare the D-KIE and other kinetic parameters at different temperatures of sLOX-1 with 13R-LOX with catalytic manganese (13R-MnLOX). The reaction rate and the D-KIE of sLOX-1 with unlabeled and [11-2H2]18:2n-6 were almost temperature independent with an apparent D-KIE of ∼56 at 30 °C, which is in agreement with previous studies. In contrast, the reaction rate of 13R-MnLOX increased 7-fold with temperature (8–50 °C), and the apparent D-KIE decreased linearly from ∼38 at 8 °C to ∼20 at 50 °C. The kinetic lag phase of 13R-MnLOX was consistently extended at low temperatures. The Phe337Ile mutant of 13R-MnLOX, which catalyzes antarafacial hydrogen abstraction and oxygenation in analogy with sLOX-1, retained the large D-KIE and its temperature-dependent reaction rate. The kinetic differences between13R-MnLOX and sLOX-1 may be due to protein dynamics, hydrogen donor–acceptor distances, and to the metal ligands, which may not equalize the 0.7 V-gap between the redox potentials of the free metals.