Spectral, Kinetic, and Thermodynamic Properties of Cu(I) and Cu(II) Binding by Methanobactin from Methylosinus trichosporium OB3b

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Abstract
To examine the potential role of methanobactin (mb) as the extracellular component of a copper acquisition system in Methylosinus trichosporium OB3b, the metal binding properties of mb were examined. Spectral (UV−visible, fluorescence, and circular dichroism), kinetic, and thermodynamic data suggested copper coordination changes at different Cu(II):mb ratios. Mb appeared to initially bind Cu(II) as a homodimer with a comparatively high copper affinity at Cu(II):mb ratios below 0.2, with a binding constant (K) greater than that of EDTA (log K = 18.8) and an approximate ΔG° of −47 kcal/mol. At Cu(II):mb ratios between 0.2 and 0.45, the K dropped to (2.6 ± 0.46) × 108 with a ΔG° of −11.46 kcal/mol followed by another K of (1.40 ± 0.21) × 106 and a ΔG° of −8.38 kcal/mol at Cu(II):mb ratios of 0.45−0.85. The kinetic and spectral changes also suggested Cu(II) was initially coordinated to the 4-thiocarbonyl-5-hydroxy imidazolate (THI) and possibly Tyr, followed by reduction to Cu(I), and then coordination of Cu(I) to 4-hydroxy-5-thiocarbonyl imidazolate (HTI) resulting in the final coordination of Cu(I) by THI and HTI. The rate constant (kobsI) of binding of Cu(II) to THI exceeded that of the stopped flow apparatus that was used, i.e., >640 s-1, whereas the coordination of copper to HTI showed a 6−8 ms lag time followed by a kobsII of 121 ± 9 s-1. Mb also solubilized and bound Cu(I) with a kobsI to THI of >640 s-1, but with a slower rate constant to HTI (kobsII = 8.27 ± 0.16 s-1), and appeared to initially bind Cu(I) as a monomer.