RE-ENGINEERING THE DIIRON SITE IN RUBRERYTHRIN TOWARDS THAT IN RIBONUCLEOTIDE REDUCTASE

Abstract In order to investigate the nature and significance of the structural differences between the diiron-oxo sites in rubrerythrin and ribonucleotide reductase R2 protein (RNR R2), two mutant rubrerythrins, E97A and L60Y, were constructed, and their X-ray crystal structures determined. In L60Y rubrerythrin the side chain of Y60 is in a similar position with respect to the diiron site as is Y122 in RNR R2. However, Y60 in rubrerythrin does not make a direct hydrogen bond to an iron-coordinating terminal carboxylate, as does Y122 in RNR R2. This difference could partly explain why no tyrosyl radical is detected in L60Y rubrerythrin, and why such a radical is formed and stabilized on Y122 in RNR R2. In E97A rubrerythrin, a second histidine, H56, has become an iron ligand, and the diiron site structure in this mutant is very similar to that in RNR R2. The iron to which H56 coordinates has moved approximately 1.6 A in E97A relative to its position in wild-type rubrerythrin. These results suggest that one of the iron atoms in the diiron site of rubrerythrin could alternate between coordination to H56 and E97, thereby facilitating either intersite electron transfer or iron uptake/release.