EPR spectroscopy of soybean lipoxygenase-1. Determination of the zero-field splitting constants of high-spin Fe(III) signals from temperature and microwave frequency dependence.

The zero-field splitting constants (D) of the different components building up the high-spin Fe(III) EPR spectrum of lipoxygenase from soybeans were determined by two methods: (1) temperature dependence studies using the low-spin Fe(III) signal of cytochrome c at g 3 for accurate measuring of the temperature in the sample; (2) by establishing g-shift upon increasing the microwave frequency. The ranges of D for the axial and rhombic species contributing to the complex signal at g 6 are found to be 1.5-3.0 K and 1.8-4.4 K, respectively. The occurrence of such large ranges is attributed to variations in amount and number of species in the different samples. The combination of the applied methods offers a more generally applicable approach to the determination of zero-field splitting constants.