Table 2. Midpoint potentials of FAD, [2Fe-2S] cluster, and the occupied/free transition at pH 7.
200 μL samples poised at varying reduction potentials were frozen with liquid nitrogen-chilled ethanol and analyzed by EPR spectroscopy. The 2-electron FAD→FADH− transition gave rise to a transient radical species at g = 2.00 that was fitted according to Hastings et al. (67). The gz = 2.03 component was fitted to the Nernst equation to determine the midpoint potential of the [2Fe-2S] cluster. EFAD-FS1 was determined by plotting the height difference between the g = 1.92 “free” component and the g = 1.91 “occupied” component as in Figure 7. The error in Em values is approximately ± 10 mV.
| FAD Em,7 (mV) | [2Fe-2S] Em,7 (mV) | EFAD-FS1 (mV) | |
|---|---|---|---|
|
| |||
| WT Sdh | −100 | −15 | −125 |
| SdhA-H45A | −187 | −15 | −195 |
| SdhA-R286A | −209 | −18 | −255 |
| SdhA-R286K | −266 | −15 | −230 |
| SdhA-R286Y | −258 | +45 | -- |
| SdhA-H242A | −202 | −10 | ND |
| SdhA-H242Y | −207 | +40 | -- |