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, and CBC-L,, 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, show ed the presence of a copper center with an EPR splitting constant between those of type 1 and type 2 copper centers (g(perpendicular to) = 2.087, g(parallel to) = 2.42 G, \A(parallel to)\ = 128 G). The EPR spectrum of CBC-L-2 was more complex and showed two spectrally distinct copper centers. One signal ran be attributed to a type 2 CU2+ center (g(perpendicular to) = 2.073, g(parallel to),= 2.324 G, \A(parallel to)\ = 144 G) which could be saturated at higher powers, while the second shows a broad, nearly isotropic signal near g(perpendicular to) = 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.