Figure 4.

Kinetic and spectroscopic evidence for state c (Fig. 2C), containing Y165•, Fe(III), and 4, in the CarC reaction. (A) Change in absorbance at the indicated reaction times after mixing the CarC•Fe(II) •2OG•3 complex with O₂. The spectra shown were obtained by subtracting the spectrum at 5 ms from the spectra at the indicated reaction times. (B) Kinetics of Y• formation and decay, as reported by the height of the sharp peak at 410 nm, in the reactions of wild-type CarC (red), and the Y67F (black), Y164F (yellow), Y165F (green), and Y191F (cyan) variants under the same conditions as in A. The orange trace is a control reaction of the wild-type enzyme from which 3 was omitted from the protein syringe. The dark blue trace is from the reaction of the wild-type enzyme in the presence of the 5-d-3 substrate. Each trace in B is the average of three trials constructed as indicated by the gold lines, bracket, and arrow in A as A₄₁₀-(A₄₀₄+A₄₁₆)/2 (A_{410 dropline}). (C) X-band EPR spectra of the Y• in reaction samples freeze-quenched at the indicated times. The 100-K spectrum of the 3-s sample (gray trace) has been scaled by 0.5 for clarity. Measurement conditions: microwave frequency, 9.4 GHz; microwave power, 2 μW for 10-K spectra and 20 μW for 100-K spectrum; modulation amplitude, 1 mT for 10-K spectra and 0.2 mT for 100-K spectrum; modulation frequency, 100 kHz. (D) Chemical-quench, LC-MS analysis of the stereoinversion of 3 to 4. Negative-mode, single-ion chromatograms at m/z = 172 for acid hydrolysis of the 3 synthetic standard (3→12, red trace) and the 3.5:1 4:3 synthetic standard (4→13, blue trace) and for CarC reactions acid-quenched at the indicated times. Reaction conditions for the four experiments, the procedures used to synthesize the standards, and details of the LC-MS analysis are provided in the Supplementary Methods.