STUDIES OF THE IRON SITE OF RIBONUCLEOTIDE REDUCTASE

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The enzyme ribonucleotide reductase (RR) catalyzes an essential step in the biosynthesis of DNA: the reduction of ribonucleotides to deoxyribonucleotides. Ribonucleotide reductase activity is essential for the survival of all cells. Non-heme iron dependent RRs can be obtained from a wide variety of sources including eucaryotic (e.g., human) cells, procaryotic cells, mammalian viruses, and bacteriophage viruses. The enzyme from E. Coli is the best characterized, and serves as a prototype for the iron dependent RRs. The E. Coli enzyme consists of two subunits, B1 and B2. The B1 subunit contains binding sites for allosteric effectors which regulate the activity and specificity of the enzyme and thereby maintain a balanced pool of deoxyribonucleotides for DNA synthesis. Perhaps the most unusual features of RR are a stable tyrosine free radical and a binuclear iron complex that are contained in the B2 subunit, and which are essential for enzyme function, and hence for DNA synthesis and replication. Very little structural information has been directly available on the binuclear Fe site, and the manner in which it generates and stabilizes the radical is not known. In this application we propose to extend our investigations of the local structures of the binuclear iron sites of the B2 subunit of E. Coli ribonucleotide reductase, the corresponding M2 subunit from the mammalian enzyme, and the holoenzyme B1 + B2 using EXAFS and XANES spectroscopy. Low noise, high energy resolution X- ray absorption spectra of native protein B2, B1 + B2, M2, oxy- hemerythrin, anaerobic apoprotein B2 incubated with ferrous iron, anaerobic deoxyhemerthrin, and suitable model compounds will be studied at 80 K, 120 K, 180 K and 300 K and will be quantitatively analyzed to determined the coordination numbers, atomic distances, and EXAFS Debye-Waller factors for the first, second and third coordination shells. From this information the Fe-O-Fe bridging angle (when the u-oxo bridge is present), the number of coordinated histidine residues, and possibly the number of bridging carboxylates will be determined. Spectra of protein B2 will also be measured at 300 K at pH 6.0, 7.0, 8.0 and 9.0, and the holoenzyme (B1 + B2) will be compared to protein B2 at 300 K pH 7.0. Other related projects include studies of the dose and dose- rate dependence of X-radiation induced damage to protein B2, and comparison of experimental XANES and EXAFS spectra to those predicted by theoretical multiple- and single-scattered wave calculations.