Figure 4.

Mössbauer spectra of double-mixing RFQ samples for reaction of the diiron(III) intermediate with buffer containing phenol. Reduced protein was allowed to react with O₂ for 0.17 s to generate the intermediate before mixing with phenol (A). After mixing with phenol, the reaction mixture was freeze-quenched at 0.14 s (B), 0.67 s (C), 5 s (D) and 100 s (E). The spectra (vertical bars) are collected at 4.2 K in a 50 mT applied field parallel to the γ-ray beam. The green, red and orange lines are simulated spectra, respectively, of the unreacted diiron(II) protein, diiron(III) intermediate and diiron(III) product. The spectra of the diiron(III) intermediate in the absence of phenol (red in A) and diiron(III) product (orange) were simulated using the parameters reported in the caption of Figure 1. The spectrum of the diiron(III) intermediate was perturbed slightly by the presence of phenol (see the following Figure 6) and was simulated with the altered parameters reported in text (red lines in B-E). The spectrum of the unreacted diiron(II) was modeled as a superposition of two quadrupole doublets with an intensity ratio of 10 to 1, and the following parameters: δ= 1.32 mm/s, ΔE_Q = 3.06 mm/s for the intense doublet, and δ = 1.39 mm/s and ΔE_Q = 2.13 mm/s for the minor doublet. The simulated spectra are plotted at the following absorption intensities: green, 61%, 60%, 58%, 57% and 29%; red, 39%, 30%, 14%, 0% and 0%; orange, 0%, 12%, 28%, 43% and 71% in A, B, C, D and E, respectively. The black lines overlaid with the spectra are composite spectra including all the Fe species mentioned above.