title: Spectral, kinetic, and thermodynamic properties of Cu(I) and Cu(II) binding by methanobactin from Methylosinus trichosporium OB3b.
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 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.
authors.affiliation: Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, Iowa 50011-3211, USA.
authors.forename: Dong W
authors.initials: DW
authors.lastname: Choi
authors.forename: Corbin J
authors.initials: CJ
authors.lastname: Zea
authors.forename: Young S
authors.initials: YS
authors.lastname: Do
authors.forename: Jeremy D
authors.initials: JD
authors.lastname: Semrau
authors.forename: William E
authors.initials: WE
authors.lastname: Antholine
authors.forename: Mark S
authors.initials: MS
authors.lastname: Hargrove
authors.forename: Nicola L
authors.initials: NL
authors.lastname: Pohl
authors.forename: Eric S
authors.initials: ES
authors.lastname: Boyd
authors.forename: G G
authors.initials: GG
authors.lastname: Geesey
authors.forename: Scott C
authors.initials: SC
authors.lastname: Hartsel
authors.forename: Peter H
authors.initials: PH
authors.lastname: Shafe
authors.forename: Marcus T
authors.initials: MT
authors.lastname: McEllistrem
authors.forename: Clint J
authors.initials: CJ
authors.lastname: Kisting
authors.forename: Damon
authors.initials: D
authors.lastname: Campbell
authors.forename: Vinay
authors.initials: V
authors.lastname: Rao
authors.forename: Arlene M
authors.initials: AM
authors.lastname: de la Mora
authors.forename: Alan A
authors.initials: AA
authors.lastname: Dispirito
mesh_terms: D002942:Circular Dichroism / Q000379:methods*; D003300:Copper / Q000737:chemistry* / Q000378:metabolism; D007093:Imidazoles / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D020602:Methylosinus trichosporium / Q000737:chemistry*; D015394:Molecular Structure; D009842:Oligopeptides / Q000737:chemistry* / Q000378:metabolism; D009942:Organometallic Compounds / Q000737:chemistry*; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods; D013052:Spectrometry, X-Ray Emission / Q000379:methods; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D013816:Thermodynamics; D013997:Time Factors
chemical_list: D007093:Imidazoles; D009842:Oligopeptides; D009942:Organometallic Compounds; C491147:methanobactin; D003300:Copper