Figure 4. Flu-TSCZ binds Cu(II) in HEPES buffer but not in YPD medium.

(a) UV-Vis spectra of Flu-TSCZ +/− Cu in 50 mM HEPES buffer, pH 7.4. Addition of Cu (blue trace) to Flu-TSCZ (purple trace) decreased the intensity of the absorption band at 321 nm and gave rise to a shoulder, indicating Cu complex formation. Addition of glycine (Gly, green trace) to the Cu(II)–Flu-TSCZ complex gave rise to a new feature at 413 nm, suggesting formation of a ternary complex, but addition of histidine (His, red trace) resulted in disappearance of the shoulder, indicating histidine competes with Flu-TSCZ for Cu(II). Conditions: [Flu-TSCZ] = 50 μM, [CuSO₄] = 25 μM, [Gly] = 1 mM, [His] = 1 mM. (b) EPR spectra of Flu-TSCZ +/− Cu in YPD medium. Addition of Cu to YPD gives rise to an EPR spectrum (solid blue trace) that is unchanged by addition of Flu-TSCZ (dotted red trace). Conditions: [Flu-TSCZ] = 300 μM, [CuSO₄] = 150 μM in YPD with 20% glycerol. Data collected at 77 K.