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HRRM2 and p53R2 are two small subunits of the ribonucleotide reductase (RR) enzyme, sharing 80% homology in amino-acid sequence with each other. RR subunits generally contribute to form RR holoenzymes for catalyzing NDP to dNDP. Here, we found another function of hRRM2 and p53R2 independent of RR enzymatic activity. Recombinant purified hRRM2 protein combined with mitochondrial extracts could dramatically induce the oxidation of 2`,7`-dichlorofluorescein (DCF) in a dose-dependent manner, while p53R2 did not. Interestingly, these two recombinant proteins showed different reactive oxygen species (ROS) EPR signal from DMPO-adduction. Site-directed mutagenesis further showed that C202Y rather than Y87F, Y176F, Y323F, and Y369F in hRRM2 destroyed hRRM2’s prooxidant property. Interestingly, Mutation of Y49F, Y138F, Y285F and Y331F rather than Y164C in p53R2 resulted in p53R2 obtaining the ability to oxidate DCF. The alteration of the oxidation of DCF did not always correspond to the changes in RR activity, in response to individual amino acid mutations. Furthermore, hRRM2 overexpression in KB cells led to a greater reduction of mitochondrial membrane potential from oxidative stress, while p53R2 overexpression did not. Moreover, overexpression of hRRM2 enhanced H2O2 induction of intracellular reactive oxygen species, while p53R2 did not. Inhibition of hRRM2 expression augmented H2O2 mediated induction of mitochondrial Mn-SOD activity. Thus, hRRM2 displays prooxidant potential, whereas p53R2 lacks this activity. On the other hand, Western blots and Confocal Microscopy demonstrated that there were high protein levels of hRRM2 and p53R2 but almost no hRRM1 in mitochondria. Thus, we proposed a model in which hRRM2 and p53R2 are not merely small subunits of the RR holoenzyme, but also might act as a depot for regulatory proteins that regulate the mitochondrial redox state. (Supported by NCI R01 Grant CA72767)

[Proc Amer Assoc Cancer Res, Volume 46, 2005]