Choi Dong W, Zea Corbin J, Do Young S, Semrau Jeremy D, Antholine William E, Hargrove Mark S, Pohl Nicola L, Boyd Eric S, Geesey G G, Hartsel Scott C, Shafe Peter H, McEllistrem Marcus T, Kisting Clint J, Campbell Damon, Rao Vinay, de la Mora Arlene M, Dispirito Alan A
Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, Iowa 50011-3211, USA.
Biochemistry. 2006 Feb 7;45(5):1442-53. doi: 10.1021/bi051815t.
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 DeltaG degrees of -47 kcal/mol. At Cu(II):mb ratios between 0.2 and 0.45, the K dropped to (2.6 +/- 0.46) x 10(8) with a DeltaG degrees of -11.46 kcal/mol followed by another K of (1.40 +/- 0.21) x 10(6) and a DeltaG degrees 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 (k(obsI)) 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 k(obsII) of 121 +/- 9 s(-)(1). Mb also solubilized and bound Cu(I) with a k(obsI) to THI of >640 s(-)(1), but with a slower rate constant to HTI (k(obsII) = 8.27 +/- 0.16 s(-)(1)), and appeared to initially bind Cu(I) as a monomer.
为了研究甲醇菌素（mb）作为甲基弯曲菌OB3b中铜获取系统细胞外成分的潜在作用，对mb的金属结合特性进行了研究。光谱（紫外可见、荧光和圆二色）、动力学和热力学数据表明，在不同的Cu（II）:mb比例下，铜的配位发生了变化。在Cu（II）:mb比例低于0.2时，mb最初似乎以同二聚体形式结合Cu（II），对铜具有较高的亲和力，结合常数（K）大于EDTA（log K = 18.8），近似ΔG°为-47 kcal/mol。在Cu（II）:mb比例为0.2至0.45之间时，K降至（2.6±0.46）×10⁸，ΔG°为-11.46 kcal/mol，随后在Cu（II）:mb比例为0.45 - 0.85时，K为（1.40±0.21）×10⁶，ΔG°为-8.38 kcal/mol。动力学和光谱变化还表明，Cu（II）最初与4 - 硫羰基 - 5 - 羟基咪唑盐（THI）以及可能的酪氨酸配位，随后还原为Cu（I），然后Cu（I）与4 - 羟基 - 5 - 硫羰基咪唑盐（HTI）配位，最终导致Cu（I）由THI和HTI配位。Cu（II）与THI结合的速率常数（k(obsI)）超过了所使用的停流装置的速率常数，即>640 s⁻¹，而铜与HTI的配位显示出6 - 8 ms的延迟时间，随后k(obsII)为121±9 s⁻¹。Mb还溶解并结合Cu（I），其与THI的k(obsI)>640 s⁻¹，但与HTI的速率常数较慢（k(obsII)=8.27±0.16 s⁻¹），并且最初似乎以单体形式结合Cu（I）。
