EPR and multi-field magnetisation of reduced forms of the binuclear iron centre in ribonucleotide reductase from mouse

Mouse ribonucleotide reductase is composed of a 1 : 1 complex of two homodimeric subunits and catalyses the first unique step on the biochemical pathway to DNA synthesis. The small subunit, protein R2, contains dinuclear iron-oxygen clusters and a tyrosyl free radical required for catalytic activity. We have studied the mixed valent and fully reduced forms of the diiron oxygen cluster from mouse R2 protein by low-temperature EPR. EPR signals of the mixed-valent states of proteins R2 reconstituted with ferrous iron and oxygen in normal and deuterated water, using the same buffers, show apparent g values of 1.92, 1.73, and 1.60 for the mixed-valent state in H2O and 1.93, 1.73, and 1.62 in D2O. These g values are typical for diiron-oxygen proteins, while the effect of D2O is unprecedented for this class of proteins. We estimate the coupling constant J for the Heisenberg exchange (H = 2J*S1*S2) to be J = –7.5±1 cm–1 for the mixed-valent form. The diferrous R2 protein shows an integer spin EPR signal in the presence of azide or 20% glycerol. Variable temperature variable field saturation magnetisation measurements show that only in the azide-complexed R2 protein does a weak ferromagnetic coupling occur (J = 0.26±0.05 cm–1), while R2 protein in the absence or presence of 20% glycerol contains non-coupled mononuclear ferrous iron (S = 2) sites.