Prev. Article Next Article Table of Contents

Article

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

Dong W. Choi,^‡ Corbin J. Zea,^§ Young S. Do,^‡ Jeremy D. Semrau, William E. Antholine, Mark S. Hargrove,^‡ Nicola L. Pohl,^§^# Eric S. Boyd,^@ G. G. Geesey,^@ Scott C. Hartsel,⁺ Peter H. Shafe,⁺ Marcus T. McEllistrem,⁺ Clint J. Kisting,^‡ Damon Campbell,⁺ Vinay Rao,⁺ Arlene M. de la Mora,^¶ and Alan A. DiSpirito*^‡

Department of Chemistry and Plant Sciences Institute, and Psychology in Education Research Lab, Iowa State University, Ames, Iowa 50011-3211, Department of Civil and Environmental Engineering, University of Michigan, Ann Arbor, Michigan 48109-2125, Department of Biophysics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, Department of Microbiology, Montana State University, Bozeman, Montana 59717, and Department of Chemistry, University of Wisconsin, Eau Claire, Wisconsin 54702

Biochemistry, 2006, 45 (5), pp 1442–1453

DOI: 10.1021/bi051815t

Publication Date (Web): January 10, 2006

†

This work was supported by Department of Energy Grant 02-96ER20237 (to A.A.D. and W.E.A.), an Inland Northwest research Alliance Graduate Fellowship grant to E.S.B., National Science Foundation Career Grant MCB 0349139 and Cottrell Awards (N.L.P.), and the Plant Sciences Institute and Department of Biochemistry, Biophysics and Molecular Biology for assistance in purchasing the isothermal titration calorimeter.

‡

Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University.

Department of Chemistry, Iowa State University.

University of Michigan.

Medical College of Wisconsin.

Plant Science Institute, Iowa State University.

Montana State University.

University of Wisconsin.

Psychology in Education Research Lab, Iowa State University.

To whom correspondence should be addressed. Telephone: (515) 294-2944. Fax: (515) 294-0453. E-mail: aland@iastate.edu.

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 (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.