Copper-Binding Compounds from Methylosinus trichosporium OB3b

Two copper-binding compounds/cofactors (CBCs) were isolated from the spent media of both the wild type and a constitutive soluble methane monooxygenase (sMMO(C)) 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-L(1) and CBC-L(2), respectively. The amino acid sequence of CBC-L(1) is S?MYPGS?M, and that of CBC-L(2) 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-L(1) 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-L(2) was more complex and showed two spectrally distinct copper centers. One signal can be attributed to a type 2 Cu(2+) 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 sMMO(C) mutants PP319 and PP359 regardless of the copper concentration in the culture medium.