Studies of cyanide binding to myeloperoxidase by electron paramagnetic resonance and magnetic circular dichroism spectroscopies

Abstract

The techniques of magnetic circular dichroism (MCD) and absorbance spectroscopy have been used to monitor titrations of oxidized myeloperoxidase with CN −, at room temperature, over the Soret, visible and near-infrared regions of the spectrum. Electron paramagnetic resonance measurements have also been made between 10–30 K. The near-infrared MCD of the unbound enzyme possesses a bi-signate band at 1000 nm, similar to those seen with other high-spin haemoproteins. On addition of CN − this band is replaced by a positive band at 1500 nm, which is assigned to low-spin haem. This switch in spin state was also observed by EPR spectroscopy with high-spin signals at g 7.09 and 5.17 titrating away to low-spin signals at g 2.58, 2.33 and 1.81. Significant changes were apparent even at ratios of CN −/haem of less than one. Integrations of the EPR spectra bave been made to check absorbance spectrum extinction coefficients, in the absorbance and MCD spectra measured over the Soret and visible bands evidence was found for more than one CN − binding process. The MCD spectrum of cyanide-bound oxidized myeloperoxidase from 350 to 700 nm strongly suggests that the haem is closely related to a ring of the chlorin type.