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.
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.
Kolberg
Matthias
M
Max-Volmer-Laboratory, Institute for Chemistry, PC 14, Technical University Berlin, D-10623 Berlin, Germany.
Logan
Derek T
DT
Bleifuss
Günther
G
Pötsch
Stephan
S
Sjöberg
Britt-Marie
BM
Gräslund
Astrid
A
Lubitz
Wolfgang
W
Lassmann
Günter
G
Lendzian
Friedhelm
F
D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D005609:Free Radicals; D008024:Ligands; D008958:Models, Molecular; D012264:Ribonucleotide Reductases / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014443:Tyrosine; D014961:X-Ray Diffraction
D029968:Escherichia coli Proteins; D005609:Free Radicals; D008024:Ligands; D014443:Tyrosine; D012264:Ribonucleotide Reductases