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J Bacteriol, July 1998, p. 3606-3613, Vol. 180, No. 14
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 (g
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Copper-Binding Compounds from Methylosinus
trichosporium OB3b
= 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.
*
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.
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