9.4 Ghz EPR Investigation of the Non-Heme Iron Active Site of Single Soybean Lipoxygenase-1 Crystals

Lipid oxidation pathways and nitric oxide signaling pathways are interrelating pathways with significance both separately and in conjunction (references 1-2). Lipid oxygenating pathways involve the formation of hydroperoxides, alkanes, alkenes, aldehydes (notably nonenal), epoxides, alcohols and other species from polyunsaturated fatty acids that are readily oxidized. The nitric oxide signaling pathway has been of great interest in the last several years and involves the formation of nitric oxide near the site of inflammation and its transport to other tissues to function as a messenger. Oxidized lipid signaling pathways are an active area of research, and many interactions with the nitric oxide pathway are left open to discussion. The interactions of nitric oxide and lipid oxidizing enzymes have been demonstrated, and these interactions are of particular significance in the regulation of vascular homeostasis. The studies presented here investigate the specific interactions of soybean lipoxygenase-1, a lipid-oxygenating enzyme, and nitric oxide by electron paramagnetic resonance (EPR) analyses of single lipoxygenase crystals complexed with nitric oxide. Nitric oxide is known to bind with high affinity to the lipoxygenase active site iron. Though nitric oxide is often used as a dioxygen analog, molecular oxygen binds to a fatty acid radical and not directly to the active site iron in the lipoxygenase mechanism, so nitric oxide was not used as a dioxygen analog in our studies. Rather, we expected the electron configuration and any differences in coordination to be a reasonable model of a transition state that mimics a peroxyl radical formed during catalysis. The aim of this project was to determine the local structure of the active site of a transition state analog with mechanistic significance. To complete this line of experimentation, I obtained a number of lipoxygenase crystals similar to those used for x-ray analysis. The crystals were then complexed with nitric oxide using protocols that are similar to those used in previous studies of lipoxygenase and other iron proteins. X-band (9.26 GHz) EPR experiments were performed and analyzed to determine the suitability of the experimental methodology presented here for observation of changes in the electronic orbital structure of the active site. Differences between the structure of the resting enzyme and the structure of the NO–LOX complex that may be observed with EPR include both the positions of the electron orbitals and the spatial orientation of the nitric oxide-iron bond, from which one may be able to infer the positions and coordination of the other iron ligands. While no conclusions about the electronic structure of the lipoxygenase iron-nitric oxide bond could be drawn from these experiments, the suitability of the experimental conditions for further studies was proven. This project also represents an advancement in the area of EPR studies of small protein crystals, similar in size to those used in x-ray diffraction experiments. Further studies of the complex that were not included in the masters project may include W-band (92.4 GHz) EPR studies and X-ray crystallography of crystals of the nitric oxide-lipoxygenase complex.