FIGURE 2.

EPR evidence for altered H/D exchange. A, EPR spectra of PFL activated by different quantities of PFL-AE and then mixed with D₂O buffer. The samples were made by fully activating 100 μm PFL and then diluting 1:1 with D₂O buffer. The solid line is 100 μm PFL activated by 100 μm PFL-AE for 30 min; dashed line is 100 μm PFL activated by 10 μm PFL-AE for 2 h; dotted line is 100 μm PFL activated by 1 μm PFL-AE for 2 h. The PFL activation mix contained 100 μm PFL, 100 μm, 10 μm, or 1 μm PFL-AE, 100 mm Tris-HCl, pH 7.6, 100 mm KCl, 500 μm AdoMet, 100 μm 5-deazariboflavin, 10 mm oxamate, and 10 mm DTT. The D₂O buffer contained 100 mm Tris-HCl, pD 8.2, and 100 mm KCl. B, reference spectra showing the PFL glycyl radical (generated by activating with 0.01 equivalent of PFL-AE) in H₂O (solid line) and after full exchange with 50% D₂O (dashed line). EPR parameters: T, 60 K; microwave frequency, 9.37 GHz; power, 19 mW; modulation amplitude, 5 G.