Lipoxygenases are an important class of non-heme iron enzymes which catalyze the hydroperoxidation of unsaturated fatty acids. This study utilizes a combination of electron paramagnetic resonance (EPR), magnetic circular dichroism (MCD), and variable-temperature, variable-field (VTVH) MCD to probe the ground state and excited states of the active site of an iron coordination mutant of soybean lipoxygenase-l (SLO-1), N694H, and relate the structural modifications with kinetic changes; The sixth coordination ligand for SLO-1, Asn(694), is proposed to be a primary factor in the coordination flexibility of the site and is reflected in both its ferrous MCD and ferric EPR spectroscopic properties. In the present study, we compare the spectroscopic properties of the N694H SLO-1 mutant with those of wild-type (WT) SLO-1 and WT human 15-lipoxygenase (15-HLO) and determine that its properties are intermediate between the WT enzymes. WT 15-HLO has a His substitution in the Asn694 equivalent position. The low-temperature MCD of the ferrous N694H SLO-1 shows that the site is 6-coordinate (6C), as in 15-HLO; however, the VTVH MCD is more similar to WT SLO-1, with a positive zero field splitting. The low-temperature EPR of ferric N694H SLO-1 exhibits a signal of intermediate rhombicity with an E/D of 0.08, which is intermediate between those for WT SLO-1 (axial, E/D approximate to 0.01) and 15-HLO (rhombic, E/D approximate to 0.33). This intermediate spectroscopic behavior can be rationalized by the stronger Ligation of the His compared to the Asn; however, since the site in WT SLO-1 is not optimized for His, it does not Ligate the iron as strongly as in the human enzyme. The H544N 15-HLO mutant was also expressed; however, it does not bind iron for activity. The kinetics of N694H SLO-1 are also intermediate (k(cat) approximate to 10 +/- 2 s(-1)) between those for SLO-1 (k(cat) approximate to 280 +/- 8 s(-1)) and HLO (k(cat) approximate to 6.2 +/- 0.1 s(-1)), which suggests that this ligand＇s interaction with the iron center may regulate the enzymatic activity. We propose that this occurs through a change in reduction potential, which in turn affects the hydrogen atom abstraction step in the reaction mechanism.