X-ray Spectroscopy of the Iron Site in Soybean Lipoxygenase-1: Changes in Coordination upon Oxidation or Addition of Methanol

Abstract

Iron K-edge X-ray spectroscopy (XANES and EXAFS) was used to study iron coordination in frozen solutions of soybean lipoxygenase-1 (SLO). The intensity of the 1s-->3d pre-edge transition of native iron(II) lipoxygenase is greater than what was found for six-coordinate high-spin iron(II) model complexes, but comparable to that of a five-coordinate model. This and a relatively short average bond length determined by EXAFS (2.13 A) indicate that the native lipoxygenase in our frozen samples is five-coordinate, excluding possible bonds longer than 2.5 A. The coordination of the iron(II) in native lipoxygenase changes 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 A, indicating a change to six-coordination. The small pre-edge feature in active iron(III) lipoxygenase suggests six-coordination. EXAFS indicates a short, 1.88 A Fe-O bond, which, given other spectroscopic and crystallographic evidence, is assigned to coordinated hydroxide. The average of the remaining bond lengths is 2.11 A. The iron coordination in iron(III) lipoxygenase is less affected by the presence of alcohols than is the site in the iron(II) enzyme. Bond valence sums indicate that the bond lengths for lipoxygenase derived from our EXAFS analyses are comparable to those of crystallographically characterized model complexes. The flexibility of the coordination number in SLON (native SLO) and the presence of an [FeIIIOH]2+ unit in SLOA (active SLO) are of possible mechanistic importance.

A Kinetic Model for the Interaction of Nitric Oxide with a Mammalian Lipoxygenase

“…Some typical examples of theoretical time courses calculated by using the parameters listed in Table 1 are shown as dotted lines in Figs 1 and 4. A rate of 200 M-' s-' for the disappearance of NO k,, was estimated from the kinetic data. Assuming an oxygen concentration of 250 pM in the aerobic assay mixture a pseudo-first-order decay k,,X[O,] of 0.05 s-' results which corresponds to a halflife time of NO of 14 s. This value is in fair agreement with NO decay rates ineasured oxygraphically in our lab (1 0 s) and with data from the literature [27]. The rate (k,) of the irreversible auto-inactivation of the enzyme varied between 62.5-163 M-' s-' for four different enzyme preparations.…”

Human deoxyhypusine hydroxylase, an enzyme involved in regulating cell growth, activates O 2 with a nonheme diiron center

“…Proposed core structure of hDOHHperoxo 9 -3 at Stanford Synchrotron Radiation Lightsource (SSRL) of the SLAC National Accelerator Laboratory and on beamline X3B at the National Synchrotron Light Source of Brookhaven National Laboratory. Standard procedures were used for data reduction, averaging, processing and EXAFS analysis using EXAFSPAK (50) and FEFF8.40 (51), preedge quantification using SSExafs (52), and XANES linear combination fitting using Athena (53). Additional details on EXAFS data collection and analysis are provided in SI Text for X-ray absorption spectroscopy, Figs.…”

A synthetic precedent for the [Fe IV 2 (μ-O) 2 ] diamond core proposed for methane monooxygenase intermediate Q

“…The monochromator was calibrated by using the edge energy of Fe foil at 7,112.0 eV. XAS data analysis was performed with the programs EXAFSPAK (43) and SSExafs (44), as previously described (45). Reactivity Studies.…”