CHARACTERIZATION OF Y122F R2 OF ESCHERICHIA-COLI RIBONUCLEOTIDE REDUCTASE BY TIME-RESOLVED PHYSICAL BIOCHEMICAL METHODS AND X-RAY CRYSTALLOGRAPHY

Ribonucleotide reductase (RNR) from Escherichia coli catalyzes the con version of ribonucleotides to deoxyribonucleotides. It is composed of two homodimeric subunits, R1 and R2. R2 contains the diferric-tyrosyl radical cofactor essential for the nucleotide reduction process. The i n vitro mechanism of assembly of this cluster starting with apo R2 or with a diferrous form of R2 has been examined by time-resolved physica l biochemical methods. An intermediate, Fe3+/Fe4+ cluster (intermediat e X), has been identified that is thought to be directly involved in t he oxidation of Y122 to the tyrosyl radical ((.)Y122). An R2 mutant in which phenylalanine has replaced Y122 has been used to accumulate int ermediate X at sufficient levels that it can be studied using a variet y of spectroscopic methods. The details of the reconstitution of the a po and diferrous forms of Y122F R3 have been examined by stopped-flow UV/vis spectroscopy and by rapid freeze quench electron paramagnetic r esonance, and Mossbauer spectroscopies. In addition the structure of t his mutant, crystallized at pH 7.6 in the absence of mercury, at 2.46 Angstrom resolution has been determined. These studies suggest that Y1 22F R2, is an appropriate model for the examination of intermediate X in the assembly process. Studies with two mutants, Y356F and double mu tant Y356F and Y122F R2, are interpreted in terms of the possible role of Y356 in the putative electron transfer reaction between the R1 and R2 subunits of this RNR.