Two active site asparagines are essential for the reaction mechanism of the class III anaerobic ribonucleotide reductase from bacteriophage T4

Class III ribonucleotide reductase is an anaerobic enzyme that uses a glycy l radical to catalyze the reduction of ribonucleotides to deoxyribonucleoti des and formate as ultimate reductant. The reaction mechanism of class III ribonucleotide reductases requires two cysteines within the active site, Cy s-79 and Cys-290 in bacteriophage T4 NrdD numbering. Cys-290 is believed to form a transient thiyl radical that initiates the reaction with substrate and Cys-79 to take part as a transient thiyl radical in later steps of the reductive reaction. The recently solved three-dimensional structure of clas s III ribonucleotide reductase (RNR) from bacteriophage T4 shows that two h ighly conserved asparagines, Asn-78 and Asn-311, are positioned close to th e essential Cys-79. We have investigated the function of Asn-78 and Asn-311 by site-directed mutagenesis and measured enzyme activity and glycyl radic al formation in five single (N78(A/C/D) and N311(A/C)) and one double (N78A /N311A) mutant proteins. Our results suggest that both asparagines are impo rtant for the catalytic mechanism of class III RNR and that one asparagine can partially compensate for the lack of the other functional group in the single Asn --> Ala mutant proteins. A plausible role for these two asparagi nes could be in positioning formate in the active site to orient it toward the proposed thiyl radical of Cys-79. This would also control the highly re active carbon dioxide radical anion form of formate within the active site before it is released as carbon dioxide. A detailed reaction scheme includi ng the function of the two asparagines and two formate molecules is propose d for class III RNRs.