Copper-Binding Compounds from Methylosinus trichosporium OB3b

Abstract

Two copper-binding compounds/cofactors (CBCs) were isolated from the spent media of both the wild type and a constitutive soluble methane monooxygenase (sMMOC) mutant, PP319 (P. A. Phelps et al., Appl. Environ. Microbiol. 58:3701–3708, 1992), of Methylosinus trichosporium OB3b. Both CBCs are small polypeptides with molecular masses of 1,218 and 779 Da for CBC-L1 and CBC-L2, respectively. The amino acid sequence of CBC-L1 is S?MYPGS?M, and that of CBC-L2 is SPMP?S. Copper-free CBCs showed absorption maxima at 204, 275, 333, and 356 with shoulders at 222 and 400 nm. Copper-containing CBCs showed a broad absorption maximum at 245 nm. The low-temperature electron paramagnetic resonance (EPR) spectra of copper-containing CBC-L1 showed the presence of a copper center with an EPR splitting constant between those of type 1 and type 2 copper centers (g⊥ = 2.087, g∥︀ = 2.42 G, ‖A∥︀‖ = 128 G). The EPR spectrum of CBC-L2was more complex and showed two spectrally distinct copper centers. One signal can be attributed to a type 2 Cu2+ center (g⊥ = 2.073, g∥︀ = 2.324 G, ‖A∥︀‖ = 144 G) which could be saturated at higher powers, while the second shows a broad, nearly isotropic signal near g⊥ = 2.063. In wild-type strains, the concentrations of CBCs in the spent media were highest in cells expressing the pMMO and stressed for copper. In contrast to wild-type strains, high concentrations of CBCs were observed in the extracellular fraction of the sMMOC mutants PP319 and PP359 regardless of the copper concentration in the culture medium.

Methanobactin and MmoD work in concert to act as the ‘copper‐switch’ in methanotrophs

“…The model proposed here is also consistent with the phenotypes of the constitutive sMMO mutants, sMMO C , isolated by Phelps and colleagues (1992). Physi- ological characterization of two sMMO C mutants, PP319 and PP359, showed these mutants constitutively expressed both methanobactin, previously called copperbinding compound, and the sMMO, regardless of the copper concentration in the growth medium (Phelps et al, 1992;DiSpirito et al, 1998). Physiological characterization of the sMMO C mutants PP319 and PP359 suggested they were both defective in the acquisition system for coppercontaining methanobactin (DiSpirito et al, 1998).…”

“…Physi- ological characterization of two sMMO C mutants, PP319 and PP359, showed these mutants constitutively expressed both methanobactin, previously called copperbinding compound, and the sMMO, regardless of the copper concentration in the growth medium (Phelps et al, 1992;DiSpirito et al, 1998). Physiological characterization of the sMMO C mutants PP319 and PP359 suggested they were both defective in the acquisition system for coppercontaining methanobactin (DiSpirito et al, 1998). The model proposed here would predict a mutation in the copper-containing methanobactin uptake system would constitutively express both methanobactin and sMMO, as observed in both PP319 and PP359.…”

Competitive ligand exchange between Cu–humic acid complexes and methanobactin

“…Most previous studies investigated methanotrophic activity and the influence of methanobactin as a response of soluble Cu concentrations (Lidstrom & Semrau, 1995;Dispirito et al, 1998). Only recently, methanotrophic behavior has been analyzed in the presence of Cu-bearing minerals (Knapp et al, 2007;Chi Fru et al, 2011;Kulczycki et al, 2011).…”

The copper responding surfaceome of Methylococccus capsulatus Bath

“…The available data suggest a significant structural diversity among the methanobactins made by methanotrophs. Methanobactin production has been demonstrated in both Gammaproteobacteria methanotrophs (M. album BG8 and M. capsulatus Bath) and Alphaproteobacteria methanotrophs (M. trichosporium OB3b and Methylocystis Strain SB2), and its production is independent on whether the cell is able to express sMMO or not (Zahn & DiSpirito, 1996;DiSpirito et al, 1998;Choi et al, 2003Choi et al, , 2005Choi et al, , 2006Choi et al, , 2008Choi et al, , 2010Kim et al, 2004Kim et al, , 2005Krentz et al, 2010). Preliminary structural characterization of the methanobactins isolated from the Gammaproteobacteria methanotrophs reveal differences from the two methanobactins that have been characterized for Alphaproteobacteria methanotrophs (Choi et al, 2010;Krentz et al, 2010).…”