Ribonucleotide reductase (RR), which reduces ribonucleotides to 2'-deoxyribonucleotides, is essential to de novo DNA synthesis, plays a direct role in regulating DNA replication, and is an attractive chemotherapeutic target. Mouse RR (mRR) is composed of two non identical subunits, mR1 and mR2; the large mR1 subunit contains both the allosteric and substrate binding sites, whereas the small mR.2 subunit contains a ?-oxo bridged diiron center which helps form and stabilize a tyrosine radical. The catalyzed reduction of substrate occurs upon a long-range electron transfer from the substrate binding site in mR1 to the tyrosine radical in the mR2. Results from our lab [(Hamann, C.S., et. al., Protein Engineering 11, 219 (1998)] have implicated the R2 C-terminus as serving a role in the electron transfer between R2 and R1. Chimeric R2 proteins (cR2) were constructed, where the last 7 or 33 residues of mR2 were substituted for the corresponding residues in eR2. When mixed with mR1, both cR2s did bind to mR1, but did not yield enzyme activity. To address this observation directly, we have constructed a ruthenium-(trisbipyridyl) linked peptide (Ru-FTLDADF), which places a photosensitizer at the N-terminus of the peptide corresponding to the seven C-terminal residues of R2 (Ac-FTLDADF is known to bind to R1, blocking not only subunit association but also enzyme activity). We intend to study the events that occur upon photoexcitation of mR1-bound Ru-FTLDADF in order to draw a conclusion on whether RuFTLDADF can substitute for the entire mR2 subunit and effect enzyme turnover.
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