FIGURE 6.

Room temperature EPR analysis of Mn²⁺ binding. A, representative room temperature continuous wave X-band EPR spectra from samples containing 100 μm of HiLpxH and the appropriate concentration of MnCl₂ to yield 1 eq of Mn²⁺, 1.4 eq of Mn²⁺, and 2.0 eq of Mn²⁺. For a comparison, an EPR spectrum from a control sample containing only buffer and MnCl₂ at the same concentration as 0.6 Mn²⁺ eq. B, double integrated intensity of free (hexoaqua) Mn²⁺ EPR signal as a function of Mn²⁺ concentration for the room temperature titration experiment (circles) and a control titration experiment without the protein present (squares). Line represents least squares fit using a two-site binding model.