AbstractThe function of ribonucleotide reductase (RNR) is to convert the four normal ribonucleotides to the corresponding deoxynucleotides. In RNR from mouse the enzyme consists of two non-identical dimers called R1 and R2, forming a heterotetramer. The R1 contains the active site with the redox active cysteines including the proposed thiyl radical site and different binding sites for allosteric effectors. The active R2 dimer harbours the essential tyrosyl radical and two m-oxo bridged diferric centres. A radical is transferred ~ 35 Å from the tyrosyl radical to the catalytic active site in the R1 forming the postulated catalytically active thiyl radical at each turnover. The thiyl radical is conserved in all three classes of RNR, but has never been observed. Neither has thiyl radicals been artificially generated in the mouse R1. Both the tyrosyl radical and the diiron centre in the active form of RNR can be reduced. The R2 subunit can exist in different redox states; among those are the active state (Tyr , Fe3+Fe3+), the mixed-valence state (Fe2+Fe3+) and the diferrous state (Fe2+Fe2+). These states are paramagnetic and can be observed by electron paramagnetic resonance (EPR). I have studied the effects of the R1 subunit on the spectroscopic properties of the tyrosyl radical and the diiron-oxygen centre in the mouse R2 subunit and chemically generated artificially novel thiyl radicals in the mouse R1 that has been studied as spin-trap adducts with EPR. A novel single turnover R1 enzyme assay was established to study the catalytically activity of the novel thiyl radicals. The novel generated thiyl radicals showed to originate in an enclosed region of the R1 protein. It proved to be more difficult to generate thiyl radicals in mouse than in E. coli and the radicals showed no catalytically activity with the single turnover enzyme. Microwave power saturation studies reveal that the half-saturation value (P1/2) for the tyrosyl radical changes more than a factor of two at 4.5 K and 13.5 K in an active holo RNR compared to the R2 alone. Furthermore, a small shift in the g1-value of the tyrosyl radical was observed with High-Field EPR (285 GHz) in the presence of R1, indicating that the neighbouring water molecule could have moved closer or changed orientation to the tyrosyl radical oxygen under turnover conditions. We could not observe any effects on the mixed-valence (Fe2+Fe3+), or on the diferrous (Fe2+Fe2+) states. Altogether, these results suggest that the tyrosyl radical is initially in a less strained electronic environment in R2 alone and that we observe for the very first time a novel protein/protein radical triggering mechanism in RNR induced by R1.