Peroxyl adduct radicals formed in the iron/oxygen reconstitution reaction of mutant ribonucleotide reductase R2 proteins from Escherichia coli

Abstract

Catalytically important free radicals in enzymes are generally formed at highly specific sites, but the specificity is often lost in point mutants where crucial residues have been changed. Among the transient free radicals earlier found in the Y122F mutant of protein R2 in Escherichia coli ribonucleotide reductase after reconstitution with Fe2+ and O2, two were identified as tryptophan radicals. A third radical has an axially symmetric EPR spectrum, and is shown here using 17O exchange and simulations of EPR spectra to be a peroxyl adduct radical. Reconstitution of other mutants of protein R2 (i.e. Y122F/W48Y and Y122F/W107Y) implicates W48 as the origin of the peroxyl adduct. The results indicate that peroxyl radicals form on primary transient radicals on surface residues such as W48, which is accessible to oxygen. However, the specificity of the reaction is not absolute since the single mutant W48Y also gives rise to a peroxyl adduct radical. We used density functional calculations to investigate residue-specific effects on hyperfine coupling constants using models of tryptophan, tyrosine, glycine and cysteine. The results indicate that any peroxyl adduct radical attached to the first three amino acid alpha-carbons gives similar 17O hyperfine coupling constants. Structural arguments and experimental results favor W48 as the major site of peroxyl adducts in the mutant Y122F. Available molecular oxygen can be considered as a spin trap for surface-located protein free radicals.

Electron-Transfer Chemistry of Ru−Linker−(Heme)-Modified Myoglobin: Rapid Intraprotein Reduction of a Photogenerated Porphyrin Cation Radical

“…The decay of the peroxo radical within 40 s is in accord with the ∼7 s lifetime of peroxo radicals in Mb . All the radicals observed in photolysis experiments on RuC7Mb, with the exception of the species corresponding to the EPR spectrum in Figure B (which persists longer than 2 min), decay within 40 s.…”

“…Horse heart myoglobin contains four aromatic amino acid residues, Y103, Y146, W7, and W14 (Figure A), whose reduction potentials should fall in the 0.6−1.0 V vs NHE − range and could, therefore, be oxidized by P •+ or Fe IV O. , We trapped protein-based radical species by photolysis/freeze-quenching of solutions containing RuC7MbM 3+ /[Co(NH 3 ) 5 Cl] 2+ , as evidenced by strong EPR signals centered at g ≈ 2.004 (Figures and ) . An intense EPR signal with Δ H pp = 15 G (Δ H pp is the peak-to-trough line width) and partially resolved hyperfine structure was observed in experiments conducted on degassed solutions containing RuC7MbFe 3+ /[Co(NH 3 ) 5 Cl] 2+ (Figure A); this spectrum features a composite signal with a substantial contribution from a neutral Y radical, possibly Y103, as this residue is very close to the heme. ,,, Formation of a peroxo radical (identified by its rhombic g tensor, g z = 2.036, g x = 2.009, g y = 2.003) when photolysis was conducted in air (Figure C) demonstrates unambiguously that W14 also is oxidized. , Upon thawing and refreezing (a process that took >40 s), a narrower (Δ H pp = 8.8 G) EPR signal having ∼20% of the intensity of the initial signal in Figure A with no sign of a peroxo species was observed (Figure B) . This EPR spectrum is significantly sharper (indicating less hyperfine coupling) 33 than those of well-characterized Y • and W • radicals, which typically have Δ H pp > 18 G, , and remains unassigned .…”

Role of Electrostatics and Salt Bridges in Stabilizing the Compound I Radical in Ascorbate Peroxidase

“…Figure 3 shows the EPR spectra for wild-type APX, APX3M, and APX3M/W179F together with the spin equivalents estimated by comparison to a Cu 2+ -EDTA standard. Previously recorded EPR spectra for aged compound I, sometimes called APX I*, showed a small g || ) 2.038 and g ⊥ ) 2.004 feature which was shown to be a Trp radical, most likely Trp179 (6,(26)(27)(28). The spectra of wild-type APX and APX3M show a similar g || ) 2.037 and g ⊥ ) 2.006 signal.…”

Spectroscopy and Reactivity of a Photogenerated Tryptophan Radical in a Structurally Defined Protein Environment

“…Density functional calculations on characterized protein radicals as well as model systems support the assignment of W108 as a neutral species; moreover, the value of g x indicates that the indole nitrogen is near an H-donor. At neutral pH, the EPR signal can be detected for over 5 h at room temperature (Figure , upper right inset); indeed, in regard to kinetic stability, the W108 radical in Re(I)AzM(II) is rivaled only by the tyrosine radicals in photosystem II (TyrD) and RNR 2 X-band EPR spectrum of ReAz(W108 • )Zn(II) under anaerobic conditions (77 K, pH 7.2 KP i , ν = 9.4753 GHz, modulation amplitude = 0.2 mT, microwave power ≈ 200 μW).…”