Near-infrared circular dichroism and magnetic circular dichroism and x-ray absorption spectral comparison of the non-heme ferrous active sites of plant and mammalian 15-lipoxygenases

Abstract

Lipoxygenases (LOs) are non-heme iron enzymes which catalyze the reaction of dioxygen with cis,cis- 1,4-pentadiene-containing fatty acids to form hydroperoxide products, which in mammals are the precursors to the inflammation- and immunity-mediating compounds lipoxins and leukotrienes. Recent X-ray crystal structures of ferrous soybean lipoxygenase-1 (SLO-I) offer two different descriptions of the active site: one four-coordinate and one five- or six-coordinate. Near-infrared (NfIR) circular and magnetic circular dichroism (CD/MCD) and variable- temperature, variable-field (VTVH) MCD have been used to study SLO-I in solution which is found to exist as a 40/60% mixture of five- and six-coordinate forms, respectively. Addition of linoleate substrate or alcohols shifts the mixture to the purely six-coordinate form. NIR CD/MCD studies of two mammalian LOs, rabbit reticulocyte and recombinant human 15-LOs, show that these exist as pure six-coordinate forms. X-ray absorption Fe K-edge and pre-edge data also show that the mammalian 15-LOs and SLO-I in glycerol are six-coordinate. This is consistent with the extended X-ray absorption fine structure (EXAFS) results of SLO-I in glycerol which show the iron active site to have 5 ± 1 N/0 at ~2.16 A. VTVH MCD data on the six-coordinate sites show that the mammalian and soybean enzymes have very different ground-state splittings, indicative of differences in bonding interactions with the ligand set. These differences in ferrous site coordination in solution and ground-state splittings are attributed to the substitution of a stronger histidine ligand in the mammalian 15-LOs for an asparagine in SLO-I.