Iron ligand mutants in protein R2 of Escherichia coli ribonucleotide reductase

Abstract

Ribonucleotide reductase protein R2 contains a diiron-oxo center with the ability to generate and stabilize a catalytically essential tyrosyl radical. The six protein-derived ligands (four carboxylates and two histidines) of the diiron site were, in separate experiments, mutated to alanines and in two cases also to histidines. We found that removal or exchange of an iron ligand did not in general abolish the formation of a diiron site in the mutant proteins, although all mutant proteins lost the bound metal ions with time upon storage. Iron bound to the mutant proteins was characterized by light absorption, EPR and resonance Raman spectroscopy. In addition, the ability of the mutant proteins to form a tyrosyl free radical and the catalytic competence of the latter were determined by EPR spectroscopy and activity measurements. The diiron sites of mutant proteins D84H and E238A were quite reminiscent of that in wild-type R2. Four of the other mutant proteins (H118A, E204A, E204H, H241A) could form the same number of metal sites as wild-type R2, but with different spectroscopic properties. The mutation E115A affecting the only μ-bridging ligand lowered the amount of bound iron to less than half. An important observation was that D84A, H118A and E204A formed transient tyrosyl radicals, but only the E204A mutant protein was enzymatically active. D84A and H118A affect iron ligands which have been suggested to participate in long-range electron transfer during catalysis. Our observation that these mutant proteins are catalytically inert, despite formation of a tyrosyl radical, underscores the necessity for an intact electron transfer pathway for catalytic activity in ribonucleotide reductase.