HPLC analysis of RCQ samples from a single-turnover reaction of S5HH. Fully reduced S5HH (256 μM), salicylate (900 μM) and internal standard caffeic acid (50 μM) were reacted with O2 (900 μM) (concentrations after mixing) in standard buffer at 4 °C. The product formation at 4 °C is largely complete before the fastest chemical quench possible (~10 ms) so an accurate rate constant could not be obtained. The dashed blue line is an exponential curve with k = 200 s−1 meant to guide the eye. Inset: EPR spectra of rapid freeze quench samples from the single-turnover reaction at the times shown. Instrument conditions: microwave power, 200 μW; modulation amplitude, 10 G; microwave frequency, 9. 644 GHz; temperature, 2 K.