Lipoxygenases catalyze reactions of polyunsaturated fatty acids with dioxygen to form lipid hydroperoxides; in humans this is the first step in production of leukotrienes from arachidonic acid, a process involved in inflammatory response. All lipoxygenases have conserved amino acid sequence regions, a single non-heme iron, and share many spectroscopic and kinetic properties. Two independent crystal structures of the inactive native form of soybean lipoxygenase establish three histidine residues and the C-terminal carboxylate as ligands to the iron(II) (Boyington, J. C.; Gaffney, B. J.; Amzel, L. M. Science 1993, 260, 1482-1486; Minor, W.; Steczko, J.; Bolin, J. T.; Otwinowski, Z.; Axelrod, B. Biochemistry 1993, 32, 6320-6323.) An asparagine side-chain carbonyl is suggested to bind iron in one structure, and both structures show a "vacant" coordination site which could be occupied by a water ligand. Our previous EXAFS analyses of frozen solutions of native lipoxygenase indicate a predominantly five-coordinate environment for the iron(II), but that this can be shifted to a six-coordinate environment by addition of 20 mM methanol, or higher concentration of glycerol, prior to freezing. As the coordination number increases, the average bond length increases by 4 pm, from 213 pm to 217 pm (Scarrow, R. C.; Trimitsis, M. G.; Buck, C. P.; Grove, G. N.; Cowling, R. A.; Nelson, M. J. Biochemistry 1994, 33, 15023-15035). We recently re-investigated the effects of methanol on the X-ray absorption spectrum of native lipoxygenase, this time in solution at 4 C. The experiments were carried out at beam line X9B using a solution cell and constant temperature bath provided by the beam line. The spectra change very little for 0 to 10% (2 M) methanol, and are all similar to the spectrum of native lipoxygenase frozen without methanol. The refined bond lengths from EXAFS are 212 =B1 1 pm. Thus the iron(II) in native soybean lipoxygenase-1 remains five-coordinate upon addition of methanol unless the sample is subsequently frozen.