Determination of the zero-field splitting constants of high-spin metalloproteins by a continuous-wave microwave saturation technique

Abstract

A method is described for determining the zero-field splitting energy of high-spin metalloenzymes and proteins by a continuous-wave saturation technique with a conventional EPR spectrometer. The method requires analysis of graphical estimates of P 1 2 from saturation curves as a function of temperature. Under the condition that the signal amplitude depends only on the spin-lattice relaxation probability, the temperature dependence of P 1 2 provides an estimate of the zero-field splitting energy for spin-lattice relaxation via an Orbach process. The zero-field splitting energies of metmyoglobin and metmyoglobin fluoride are estimated at 22 and 12 cm −1, respectively, in excellent agreement with published results obtained by magnetic susceptibility and pulse saturation recovery methods. The zero-field splitting of methemoglobin is sensitive to the binding of inositol hexaphosphate. This change is shown to be consistent with the change in iron-porphyrin stereochemistry induced by binding of the organic phosphate molecule to the protein. The method can be applied to a wide variety of high-spin paramagnetic systems.