A new tyrosyl radical on Phe(208) as ligand to the diiron center in Escherichia coli ribonucleotide reductase, mutant R2-Y122H - Combined X-ray diffraction and EPR/ENDOR studies
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The R2 protein subunit of class I ribonucleotide reductase (RNR) belongs to a structurally related family of oxygen bridged diiron proteins. In wild-type R2 of Escherichia coli, reductive cleavage of molecular oxygen by the diferrous iron center generates a radical on a nearby tyrosine residue (Tyr122), which is essential for the enzymatic activity of RNR, converting ribonucleotides into deoxyribonucleotides. In this work, we characterize the mutant E. coli protein R2-Y122H, where the radical site is substituted with a histidine residue. The x-ray structure verifies the mutation. R2-Y122H contains a novel stable paramagnetic center which we name H, and which we have previously proposed to be a diferric iron center with a strongly coupled radical, (FeFeR)-Fe-III-R-III . Here we report a detailed characterization of center H, using H-1/H-2-N-14/N-15- and Fe-57-ENDOR in comparison with the (FeFeIV)-Fe-III intermediate X observed in the iron reconstitution reaction of R2. Specific deuterium labeling of phenylalanine residues reveals that the radical results from a phenylalanine. As Phe(208) is the only phenylalanine in the ligand sphere of the iron site, and generation of a phenyl radical requires a very high oxidation potential, we propose that in Y122H residue Phe(208) is hydroxylated, as observed earlier in another mutant (R2-Y122F/E238A), and further oxidized to a phenoxyl radical, which is coordinated to Fe1. This work demonstrates that small structural changes can redirect the reactivity of the diiron site, leading to oxygenation of a hydrocarbon, as observed in the structurally similar methane monoxygenase, and beyond, to formation of a stable iron-coordinated radical.
|Research areas and keywords||
Subject classification (UKÄ) – MANDATORY
|Journal||Journal of Biological Chemistry|
|Publication status||Published - 2005|