FIGURE 9. KmtR has tighter affinity for nickel and cobalt than NmtR.

A, Ni(II) binding isotherm for KmtR (5 μm) monitored as tryptophan fluorescence (λ_ex = 280 nm, λ_em = 440 nm). K_Ni is too tight to measure under these conditions. B, competition between KmtR (5 μm) and NmtR (5 μm) for Ni(II) (4 μm) monitored as tryptophan emission spectra following excitation at 295 nm. Only KmtR contains a tryptophan residue. Ni(II)-dependent difference spectra are shown for KmtR (5 μm) alone (open circles) and KmtR in the presence of equimolar NmtR (closed circles). The lower curve (triangles) shows the negligible Ni(II)-dependent difference spectrum for tyrosine residues of NmtR (5 μm) alone at this excitation wavelength. C, Co(II) binding isotherm for KmtR monitored as tryptophan fluorescence (λ_ex = 280 nm, λ_em = 440 nm). The curve represents a 1:1 ligand binding model, which under these conditions (including 1 mm DTT) gives K_KmtR = 6.9 μm. K_NmtR under these conditions is estimated to be weaker than K_KmtR and weaker than we reported previously in the absence of DTT (13). D, competition between KmtR (5 μm) and NmtR (5 μm) for Co(II) (4 μm) monitored as tryptophan emission spectra following excitation at 295 nm. Co(II)-dependent difference spectra are shown for KmtR (5 μm) alone (open circles) and KmtR (5 μm) in the presence of equimolar NmtR (closed circles). The lower curve (triangles) shows the negligible Co(II)-dependent difference spectrum for tyrosine residues of NmtR (5 μm) alone at this excitation wavelength. Conditions are 20 mm HEPES, 1 mm DTT, 50 mm NaCl (pH 7.5), 22 °C.