X-Ray Absorption Studies into the iron Ligand Sphere of Plant and Animal Lipoxygenases

Abstract

Lipoxygenases (LO’s) constitute a family of lipid peroxidizing enzymes which are classified according to their positional specificity of arachidonic acid oxygenation (Kuhn, 1996). In plants LO’s are known for a long time. Some 60 years ago a lipoxidase was identified in dry soybeans. This enzyme and other plant LO’s were later on purified and characterized with respect to their protein chemical and enzymatic properties (Siedow, 1991). For a long time it was believed that LO’s are restricted to the plant kingdom but along with the advances in eicosanoid research LO’s have been discovered in the animal kingdom. In 1975 an arachidonate 15-LO was detected in rabbit reticulocytes which is high level expressed in these immature red blood cells. Since then a variety of LO’s isoforms have been described in plants and animals and our knowledge on the enzymology and molecular biology of LO’s has significantly increased during the past 15 years. However, direct structural information on LO’s is still somewhat limited. LO’s contain one gram-atom non-heme iron per mole enzyme. In 1993 two independent low resolution crystal structures for the soybean LO-1 were published (Boyington et al., 1993, Minor et al., 1993). Later on high resolution crystal structures were reported for two soybean LO isoenzymes (Minor et al., 1996; Skrzypczak-Jankun et al., 1997). More recently, the crystal structure of a rabbit reticulocyte 15-LOX/inhibitor complex was solved (Gillmor et al., 1997). In addition to these crystallographic studies there is a number of spectroscopic investigations aimed to determine the geometry of the iron ligand sphere. Investigations of the magnetic susceptibility (Petersson et al., 1985) suggested that the ferrous active site ground state is high spin with S=2. Measurements of the X-ray absorption (van der Heijdt et al., 1992; Scarrow, 1994), Mossbauer spectroscopy (Dunham, et al., 1990) and experiments on the magnetic circular dichroism (Whittaker and Solomon, 1988) predicted a distorted octahedral iron ligand sphere for plant LO’s. Studies of the circular dichroism as well as evaluation of the ls-3d transition pre-edge signal in the X-ray absorption spectra suggested that plant and mammalian 15-LO’s in their non-activated ground state may contain 6-coordinate ferrous iron (Pavlosky et al., 1995). Site directed mutagenesis studies suggested 4 histidines and the C-terminal isoleucine as iron liganding amino acids of various mammalian LO’s and the sixth ligand was supposed to be a water molecule (Kuban et al., 1998). In the soybean LO-1 one iron liganding histidine is substituted for by an asparagine, and here again a water oxygen was identified as sixth iron ligand. In a recent review article new insights into the structure/function relationship of LO’s are provided (Solomon, 1997).