X-RAY SPECTROSCOPY OF THE IRON SITE IN SOYBEAN LIPOXYGENASE-1 - CHANGES IN COORDINATION UPON OXIDATION OR ADDITION OF METHANOL

Iron K-edge X-ray spectroscopy (XANES and EXAFS) was used to study iro n coordination in frozen solutions of soybean lipoxygenase-1 (SLO). Th e intensity of the 1a-->3d pre-edge transition of native iron(II) lipo xygenase is greater than what was found for six-coordinate high-spin i ron(II) model complexes, but comparable to that of a five-coordinate m odel. This and a relatively short average bond length determined by EX AFS (2.13 Angstrom) indicate that the native lipoxygenase in our froze n samples is five-coordinate, excluding possible bonds longer than 2.5 Angstrom. The coordination of the iron(II) in native lipoxygenase cha nges when methanol (as low as 0.1%) or glycerol (20%) is added to the buffer prior to freezing. The addition of methanol diminishes the pre- edge transition and increases EXAFS-derived bond lengths by 0.04 Angst rom, indicating a change to six-coordination. The small pre-edge featu re in active iron(III) lipoxygenase suggests six-coordination. EXAFS i ndicates a short, 1.88 Angstrom Fe-O bond, which, given other spectros copic and crystallographic evidence, is assigned to coordinated hydrox ide. The average of the remaining bond lengths is 2.11 Angstrom. The i ron coordination in iron(III) lipoxygenase is less affected by the pre sence of alcohols than is the site in the iron(II) enzyme. Bond valenc e sums indicate that the bond lengths for lipoxygenase derived from ou r EXAFS analyses are comparable to those of crystallographically chara cterized model complexes. The flexibility of the coordination number i n SLO(N) (native SLO) and the presence of an [(FeOH)-O-III](2+) unit i n SLO(A) (active SLO) are of possible mechanistic importance.