A New Tyrosyl Radical on Phe super(208) as Ligand to the Diiron Center in Escherichia coli Ribonucleotide Reductase, Mutant R2-Y122H: Combined X-ray diffraction and EPR/ENDOR studies

• Published in: The Journal of biological chemistry 2005-03, Vol.280 (12), p.11233-11246
• Main Authors: Kolberg, Matthias, Logan, Derek T, Bleifuss, Gunther, Poetsch, Stephan, Sjoeberg, Britt-Marie, Graeslund, Astrid, Lubitz, Wolfgang, Lassmann, Gunter, Lendzian, Friedhelm
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X

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 (Tyr super(122)), 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, Fe super(III)Fe super(III)R.. Here we report a detailed characterization of center H, using super(1)H/ super(2)H- super(14)N/ super(15)N-and super(57)Fe-ENDOR in comparison with the Fe super(III)Fe super(IV) 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 super(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 super(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.