A new tyrosyl radical on Phe208 as ligand to the diiron center in Escherichia coli ribonucleotide reductase, mutant R2-Y122H. Combined x-ray diffraction and EPR/ENDOR studies.
2005 (English)In: J Biol Chem, ISSN 0021-9258, Vol. 280, no 12, 11233-46 p.Article in journal (Other academic) Published
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, Fe(III)Fe(III)R.. Here we report a detailed characterization of center H, using 1H/2H -14N/15N- and 57Fe-ENDOR in comparison with the Fe(III)Fe(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 Phe208 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 Phe208 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.
Place, publisher, year, edition, pages
2005. Vol. 280, no 12, 11233-46 p.
Electron Spin Resonance Spectroscopy, Escherichia coli/*enzymology, Escherichia coli Proteins/*chemistry, Free Radicals, Ligands, Models; Molecular, Ribonucleotide Reductases/*chemistry, Spectrometry; Mass; Matrix-Assisted Laser Desorption-Ionization, Tyrosine, X-Ray Diffraction
IdentifiersURN: urn:nbn:se:su:diva-20342PubMedID: 15634667OAI: oai:DiVA.org:su-20342DiVA: diva2:186868