FIGURE 4.

EPR spectroscopy and redox titration of the diiron site. WT EhFdp1 (50 μm) in 50 mm Tris-HCl, 18% (v/v) glycerol, pH 7.5, was anaerobically redox titrated, by stepwise addition of sodium dithionite. EPR spectra were recorded at 7 K. Panel A, spectra recorded along the titration, with the corresponding redox potentials. Panel B, titration profile of the WT EhFdp1 mixed valence diiron center; dots represent normalized intensities (heights relative to the maximal values) measured at g = 1.95 and 1.77; full line represents the best adjustment using a Nernst equation for two sequential monoelectronic reduction steps, with E₁ = +170 mV and E₂ = +132 mV. Panel C, EPR spectra of the mixed valence diiron center in WT and mutated EhFdp1. Microwave frequency: 9.39 GHz; microwave power, 2 milliwatt; modulation amplitude, 1 millitesla.