Characterization of the paramagnetic centres of the molybdenum-iron protein of nitrogenase from Klebsiella pneumoniae using low temperature magnetic circular dichroism spectroscopy

The magnetic circular dichroism (MCD) spectra of the dithionite-reduced and thionine-oxidized forms of the molybdenum-iron protein, Kp1, from the nitrogenase of Klebsiella pneumoniae have been recorded between 300–1000 nm at temperatures from 1.5 to 120 K and at magnetic fields up to 5.1 T. At these temperatures the MCD spectrum is dominated by the contributions from the paramagnetic centres present. Therefore, the low-temperature MCD spectrum of dithionite-reduced Kp1 arises from the EPR-active molybdenum-iron centre known to be the cofactor extractable with $\text{N-}\text{methylformamide}$. The MCD spectra of thionine-oxidized Kp1 reveal the form of the optical spectrum of the other major iron-containing components in the protein, which are believed to be in iron-sulphur clusters. MCD magnetization curves are quite distinctive for the two types of paramagnet. The curve for the oxidized form of Kp1 shows the unusual magnetic properties of the EPR-silent paramagnetic centre. It has an electronic doublet as the lowest component of a complex ground state. At 1.54 K this centre magnetizes steeply with increase in magnetic field such that it is more than 90% magnetized at 1.5 T. The spin of the cluster is estimated to be either $\text{7}\text{2}$ or $\text{5}\text{2}$. The forms of the MCD magnetization curves and of the MCD spectra provide an excellent set of spectroscopic fingerprints for the two predominant types of metal centre. Comparison of these properties with those of model compounds, with extracted cofactor and with apoprotein should provide criteria for assessing the validity of structural models for the complex metal centres in nitrogenase.