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 (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-L2 was 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.
DiSpirito
A A
AA
Department of Microbiology, Immunology, and Preventive Medicine, Iowa State University, Ames, Iowa 50011, USA. aland@aistate.edu
Zahn
J A
JA
Graham
D W
DW
Kim
H J
HJ
Larive
C K
CK
Derrick
T S
TS
Cox
C D
CD
Taylor
A
A
D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D003300:Copper / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008772:Methylococcaceae / Q000378:metabolism*; D009154:Mutation; D013057:Spectrum Analysis
D001426:Bacterial Proteins; D002352:Carrier Proteins; C023548:copper-binding protein; D003300:Copper