Long range radical initiation essential for class I ribonucleotide reductases: Direct evidence for amino acid radical intermediates

ORGN 659

JoAnne Stubbe, Stubbe@mit.edu, Massachusetts Institute of Technology, MA, Mohammad Seyedsiamdost, Department of Chemistry and Biology, MIT, 77 Massachusetts Ave, Cambridge, MA 02139, and Daniel G. Nocera, nocera@mit.edu, Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, 6-335, Cambridge, MA 02139.
Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms and thus play a central role in DNA replication and repair. The Class I RNRs have a diferric-tyrosyl radical (Y•) cofactor on the R2 subunit that catalyzes thiyl radical formation on the R1 subunit 35 Å away. The radical initiation process has been proposed to involve amino acid radical intermediates and a pathway: Y122 to W48 to Y356 (within R2) to Y731 to Y730 and C439 within R1. We have developed methods to synthesize R2 semisynthetically using intein chemistry. Y356 has been replaced by 2,3 F2Y, 3,5 F2Y, 2,3,5 F3Y, 2,3,6 F3Y, F4Y and dopa using this technology. By changes in pH and redox potential, the rate limiting step can change from a physical step to electron transfer (ET), allowing interrogation of the ET process. Studies provide direct support for a redox active Y356 in which nucleotide reduction can be turned off by 100 mv change in reduction potential. The Dopa analog at Y356 allows trapping of the hole and detection by Vis spectroscopy and EPR. Insights about the unusual radical initiation process will be presented.
 

Nakanishi Prize
1:00 PM-5:00 PM, Wednesday, 16 March 2005 Convention Center -- Ballroom 20A-B, Oral

Division of Organic Chemistry

The 229th ACS National Meeting, in San Diego, CA, March 13-17, 2005