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J Bacteriol, July 1998, p. 3606-3613, Vol. 180, No. 14
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Copper-Binding Compounds from Methylosinus trichosporium OB3b

Alan A. DiSpirito,1,* James A. Zahn,1 David W. Graham,2 Hyung J. Kim,2 Cynthia K. Larive,3 Tiffany S. Derrick,3 Charles D. Cox,4 and Alan Taylor5

Department of Microbiology, Immunology, and Preventive Medicine, Iowa State University, Ames, Iowa 500111; Department of Civil and Environmental Engineering2 and Department of Chemistry,3 University of Kansas, Lawrence, Kansas 66045; Department of Microbiology, University of Iowa, Iowa City, Iowa 522424; and Department of Biological Sciences, Wichita State University, Wichita, Kansas 672605

Received 12 September 1997/Accepted 11 May 1998

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 (gperp  = 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 (gperp  = 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 gperp  = 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.

* Corresponding author. Mailing address: Department of Microbiology, Immunology and Preventive Medicine, Iowa State University, 207 Science Building I, Ames, IA 50011-3211. Phone: (515) 294-2944. Fax: (515) 294-6019. E-mail: aland{at}iastate.edu.

J Bacteriol, July 1998, p. 3606-3613, Vol. 180, No. 14
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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