Rational reprogramming of the R2 subunit of Escherichia coli ribonucleotide reductase into a self-hydroxylating monooxygenase

The outcome of O-2 activation at the diiron(II) cluster in the R2 subunit o f Escherichia coli (class I) ribonucleotide reductase has been rationally a ltered from the normal tyrosyl radical (Y122)(1) production to self-hydroxy lation of a phenylalanine side-chain by two amino acid substitutions that l eave intact the (histidine)(2)-(carboxylate)(4) ligand set characteristic o f the diiron-carboxylate family. Iron ligand Asp (D) 84 was replaced with G lu (E), the amino acid found in the cognate position of the structurally si milar diiron-carboxylate protein, methane monooxygenase hydroxylase (MMOH). We previously showed that this substitution allows accumulation of a mu -1 ,2-peroxodiiron(III) intermediate,(2 3) which does not accumulate in the wi ld-type (wt) protein and is probably a structural homologue of intermediate P (H-peroxo) in O-2 activation by MMOH.(4) In addition, the near-surface r esidue Trp (W) 48 was replaced with Phe (F), blocking transfer of the "extr a" electron that occurs in wt R2 during formation of the formally Fe(LII)Fe (IV) cluster X.(5-7) Decay of the mu1,2-peroxodiiron(III) complex in R2-W38 F/D84E gives an initial brown product, which contains very little YI22(.) a nd which converts very slowly (t(1/2) similar to 7 h) upon incubation at 0 degreesC to an intensely purple final product. X-ray crystallographic analy sis of the purple product indicates that F208 has undergone epsilon -hydrox ylation and the resulting phenol has shifted significantly to become st lig and to Fe2 of the diiron cluster. Resonance Raman (RR) spectra of the purpl e product generated with O-16(2) or O-18(2) show appropriate isotopic sensi tivity in bands assigned to O-phenyl and Fe-O-phenyl vibrational modes, con firming that the oxygen of the Fe(III)-phenolate species is derived from Or . Chemical analysis, experiments involving interception of the hydroxylatin g intermediate with exogenous reductant, and Mossbauer and EXAFS characteri zation of the brown and purple species establish that F208 hydroxylation oc curs during decay of the peroxo complex and formation of the initial brown product. The slow transition to the purple Fe(LII)-phenolate species is asc ribed to a ligand rearrangement in which mu -O2- is lost and the F208-deriv ed phenolate coordinates. The reprogramming to F208 monooxygenase requires both amino acid substitutions, as very little epsilon -hydroxyphenylalanine is formed and pathways leading to Y122(.) formation predominate in both R2 -D84E and R2-W48F(2-7).