Spectroscopic characterization of W227F intermediate C. The 406.7 nm-excited RR of ferric DaCld(W227F) reacted with PAA at 20 °C. Initial ferric spectra are in black for pH 7.9 and blue for pH 5.8. Spectra of intermediate C at pH 7.9 (red) and pH 5.8 (green) were obtained with addition of 18 and 10 equivalents of PAA, respectively. Each intermediate C spectrum represents 15 s reaction times, Inset: The 9-GHz EPR spectrum of the protein-based radical in W227F at pH 7.9 reacted with 18 equivalents PAA for 20 s. Experimental conditions: temperature, 77 K; modulation amplitude, 4.0 G; microwave power, 2.0 mW; modulation frequency, 100 kHz.