THE INITIAL CHARACTERIZATION OF THE IRON ENVIRONMENT IN LIPOXYGENASE BY MOSSBAUER-SPECTROSCOPY

The incorporation of 57Fe into two lipoxygenase isoenzymes from soybeans has been achieved making possible the first observations of the iron environment in these proteins using Mössbauer spectroscopy. Immature soybean seeds were grown in tissue culture medium supplied with 57Fe. The iron in the active lipoxygenases that were isolated from the cultured seeds was readily detected in Mössbauer measurements. It was unequivocally demonstrated that the native enzyme contains high‐spin Fe(II). Based on the sign of the electric field gradient, the most likely ligand sphere for the iron in native lipoxygenase consists of oxygen and nitrogen ligands in a roughly octahedral field of symmetry. It was possible to detect Mössbauer signals in highly concentrated samples of native lipoxygenases containing 57Fe at natural abundance. The spectra obtained for enriched and natural abundance native enzyme had the same high‐spin Fe(II) Mössbauer parameters. This confirmed that the environment of the iron in enzymes isolated from cultured seeds and dry soybeans were the same. The Mössbauer spectra (4.2–250 K) for samples of both isoenzymes after oxidation of the iron in native enzyme by the product of lipoxygenase catalysis were extremely broad (20 mm s) with no obvious narrow resonance lines. This was the result of the existence of paramagnetically broadened spectra for such samples even at relatively high temperature as evidenced by the appropriate EPR signal. A small molecule containing an iron site sharing many of these Mössbauer and electron paramagnetic resonance properties with lipoxygenase was identified: Fe(II)/(III) · diethylenetriaminepentaacetic acid. Copyright © 1990, Wiley Blackwell. All rights reserved