Figure 4.

FIH catalysed His-hydroxylation occurs at the β-position. Hydroxylated TNKS2 538–558 peptide (RVSVVEYLLQHGADVHAKDKG) was produced by incubation with FIH under standard assay conditions (hydroxylated to ∼ 75% as assessed by MALDI-TOF analyses), LC-MS purified and analysed by NMR spectroscopy. (B) ¹H NMR spectrum of the hydroxylated TNKS2 538–558 peptide in ²H₂O. The resonances at 4.87 and 5.50 ppm, which are absent in the ¹H NMR spectrum of the nonhydroxylated TNKS2 538–558 peptide (A), are ascribed to the α- and β-proton, respectively, of the hydroxylated His residues. The resonances for the imidazole ring protons (at positions 2 and 5) are deshielded in the spectrum of the hydroxylated peptide compared to that of the nonhydroxylated. (C) 2D ¹H–¹H COSY spectrum of the hydroxylated TNKS2 538–558 peptide in ²H₂O indicating the ¹H–¹H correlation between resonances arising from the α- and β-hydrogens of the hydroxylated His residue.