Fig. 2: Substrate-triggered O2 activation by PolF.
From: Azetidine amino acid biosynthesis by non-haem iron-dependent enzymes
a, Absorption spectra acquired after a rapid mixing at 5 °C of an anoxic solution of 0.3 mM PolF, 1 mM l-Ile and 0.6 mM Fe(II) with an equal volume of O2-saturated buffer. b,c, Stopped-flow traces monitored at 614 nm. The reaction conditions were identical to a except that the substrate (l-Ile (b) or l-Val (c)) concentration was varied as shown in the figures. Each trace was fit to equation (2) to determine the rate constants summarized in Supplementary Table 4. d, Mössbauer spectra of the PolF reaction using l-Val as a substrate. Spectra were acquired at 4.2 K in a 53-mT magnetic field externally applied parallel to the propagation direction of the γ beam. The experimental spectra are depicted by vertical bars of heights reflecting the standard deviations of the absorption values during the acquisition of the spectra. Spectrum (i) is an anoxic solution of the reactant complexes (1.44 mM PolF, 2.88 mM 57Fe(II) and 40 mM l-Val). Spectra (ii)–(iv) are the reactions initiated by mixing the anoxic solution with O2-saturated buffer and incubated at 5 °C for the time shown in the figure. Shown at the bottom is the difference spectra (iii–i) overlaid with their simulations with quadrupole doublets, demonstrating the formation of μ-peroxo-Fe(III)2 (blue trace δ = 0.58 mm s−1 and ΔEQ = 1.26 mm s−1) at the expense of the consumption of high-spin Fe(II)2 (red trace δ = 1.23 mm s−1 and ΔEQ = 2.65 mm s−1). Source data for a, b, c are provided.
