FIGURE 7.
Comparison of the free radical EPR signals in different variants of DHP A and DHP B. The tyrosyl radicals mostly responsible for the line shapes presented in different panels are indicated in small boxes (see “Discussion” for these assignments). A, the DHP A signal previously obtained as the difference indicated and attributed to the Tyr-38 radical (pH 5 radical) (40) and the EPR spectrum detected in the Y28F mutant of DHP B. The two EPR spectra were obtained on different EPR spectrometers operating at slightly different microwave frequencies causing the same EPR signal to appear at slightly different magnetic field values. Therefore, the absolute values of the magnetic field are not indicated, and the signals were superimposed on the basis of the g-factors. The positions of the eight components of the EPR signal, common for both spectra, are indicated. B, the EPR signals of the free radicals formed in DHP A (Y34F/Y38F) and DHP B (Y38F). (note: DHP B does not have Tyr-34). The third signal of a similar line shape is from WT DHP B but freeze-quenched at a later time after the addition of peroxide (37). The bottom spectrum is a simulation of the tyrosyl radical EPR spectrum for the TRSSA (41) input parameters ρC1 = 0.38 and θ = 46.0°; the simulation parameters yielded by this two-value input are given in Table 4. C, the EPR spectra of the Tyr-34 radical in WT DHP A (as reported in Ref. 40) and in the DHP A Y38F mutant (as in Fig. 4). Similar to panel A, the two spectra were measured on different EPR spectrometers and the magnetic field values are not shown. D, EPR spectra show the presence of the Tyr-38 radical in WT DHP B (as a major species) (37), in WT DHP A (as a minor species but seen well at pH 5) (40) and in the Y34F mutant of DHP A at pH 7 (as in Fig. 4). Note that the free radical spectrum of WT DHP B at a later time point (see panel B) is consistent with the radical being localized on a different residue, Tyr-28. As in A and C, the magnetic field values are not shown.