Figure 6.

Rapid freeze-quench-derived EPR spectra of the products of the pre-steady-state PHM reaction. EPR spectra were generated by rapidly mixing fully reduced ascorbate-free PHM (400 mM in Cu(I)) with the oxygenated buffer containing equimolar dansyl-YVG and freezing in liquid ethane (−120 °C). The top panel shows the actual spectra, and the bottom panel shows the quantitation of the Cu(II) signal by reference to a standard Cu(II)–EDTA sample: red, 13 ms; green, 25 ms; blue, 100 ms; orange, 300 ms; and purple, 250 μM Cu(II)–EDTA standard. To avoid errors from integration of noise peaks, intensities were determined from peak-to-peak heights at g⊥ relative to that of a 250 μM solution of Cu(II)–EDTA taken through an identical cycle of mixing, freezing, and packing. Errors in concentrations determined from the peak heights are estimated from triplicate measurements of a Cu(II)–EDTA standard to be ≅ 15% and are mainly due to differences in the packing density of the samples. EPR conditions: frequency 9.63 GHz, T = 100 K, microwave power 20 mW, gain 10 dB, modulation amplitude 10 G, and sweep time 84 s.