Copper-Binding Compounds from <em>Methylosinus trichosporium</em> OB3b

A DiSpirito

J Zahn

D Graham

H Kim

Department of Microbiology, Immunology, and Preventive Medicine, Iowa State University, Ames, Iowa 50011^{; Department of Civil and Environmental Engineering}^{and Department of Chemistry,}^{University of Kansas, Lawrence, Kansas 66045; Department of Microbiology, University of Iowa, Iowa City, Iowa 52242}^{; and Department of Biological Sciences, Wichita State University, Wichita, Kansas 67260}⁵

^{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:}ude.etatsai@dnala.

Received 1997 Sep 12; Accepted 1998 May 11.

Abstract

Two copper-binding compounds/cofactors (CBCs) were isolated from the spent media of both the wild type and a constitutive soluble methane monooxygenase (sMMO^{) 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 Cu^{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^{mutants PP319 and PP359 regardless of the copper concentration in the culture medium.}

Abstract

In methanotrophs, the relationship between the concentration of copper and expression of the two different methane monooxygenases (MMOs) is well characterized (8, 11, 45, 49, 50). Under low copper-to-biomass ratios, methane oxidation activity is observed in the soluble fraction, and the enzyme is referred to as the soluble methane monooxygenase (sMMO). At higher copper-to-biomass ratios, methane oxidation activity is observed in the membrane fraction, and the enzyme is referred to as the membrane-associated or particulate methane monooxygenase (pMMO). The polypeptides and structural genes for both enzymes have been characterized (4, 18–22, 24, 25, 32, 34–40, 43–45, 47–49, 51, 62, 63). In addition to expression of the two MMOs, four other physiological traits have been identified in cells expressing the pMMO that are affected by the copper concentration in the culture medium. First, the concentration of copper in the culture media is directly related to pMMO activity in cell-free fractions, although the levels of expression of pMMO polypeptides vary in different methanotrophs (1, 8, 30, 36, 50, 63). For example, the expression levels of the three pMMO polypeptides in Methylococcus capsulatus Bath remained constant with varying copper concentrations (8, 36), whereas in Methylomicrobium albus BG8, the expression level of the putative pMMO polypeptides increased with increased copper in the culture medium (8). Second, the concentrations of membrane-associated copper and iron show a proportional increase as the copper concentration in the culture medium is increased (36, 63). Third, the formation and level of intracytoplasmic membranes in cells cultured in copper-supplemented media are dependent on the copper concentration in the culture media (8, 11, 40, 48). Lastly, the K_s for methane oxidation by pMMO is altered by the copper concentration in the culture media (33a).

Berson and Lidstrom (1) have recently noted that in spite of the central role of copper in the physiology of methanotrophs, the mechanism(s) of copper acquisition remains vague. Although true, a few studies have suggested the existence of a specific copper acquisition system in M. capsulatus Bath and M. trichosporium OB3b. The first indication of a specific copper uptake system was provided from phenotypic characterization of the constitutive sMMO mutants (sMMO^{) isolated by Phelps et al. (}42). Fitch et al. (17) found that in M. trichosporium OB3b, these sMMO^{mutants were defective in copper uptake and showed preliminary evidence for an extracellular copper-complexing agent. Working with the same mutants, Téllez et al. partially purified this copper-complexing agent and determined that it was a small molecule with a molecular mass of approximately 500 Da with an association constant with copper of 1.4 × 10}^M⁽55). Other evidence for a specific copper uptake system was provided by the copper-binding cofactor (CBC) from M. capsulatus Bath (63). During the isolation of the pMMO from M. capsulatus Bath, CBC was identified in association with the purified enzyme, in the washed membrane fraction, and in the extracellular fraction. The CBC was determined to be a small polypeptide with a molecular mass of 1,232 Da. In M. capsulatus Bath, the cellular location of the CBC varied depending on the copper concentration in the culture medium and on the expression of the pMMO.

This paper ties together and extends these observations on specific copper acquisition systems in M. trichosporium OB3b and M. capsulatus Bath. Here we describe the initial isolation and characterization of two copper-complexing agents, called CBC-L₁ and CBC-L₂, from the M. trichosporium OB3b wild type and sMMO^{mutant PP319. CBC-L}₁ from M. trichosporium OB3b was identical to the CBC previously identified during the isolation of the pMMO from M. capsulatus Bath. This paper is also the first report of a second CBC, CBC-L₂, which may have been present as a contaminant in previous CBC preparations from M. capsulatus Bath. One or both of the CBCs appear to be the same copper-complexing agent partially purified by Téllez et al. (55). Lastly, this report describes the effect of the copper concentration in the culture medium on copper uptake, the expression of both MMOs, and extracellular concentration of the CBC in wild-type and sMMO^{mutant strains of}M. trichosporium OB3b.

ACKNOWLEDGMENTS

We thank A. B. Hooper (University of Minnesota), J. D. Semrau (University of Michigan), and D. J. Thiele (University of Michigan) for useful discussions, R. Arnold (University of Arizona) for providing unpublished data, R. S. Hanson (University of Minnesota) for antibodies to the reductase and B subunits of M. trichosporium OB3b sMMO, and J. Nott (ISU Protein Facility) for technical assistance.

This work was supported by Department of Energy 02-96ER20237 (A.A.D.), the Iowa State University Office of Biotechnology (A.A.D.), an Iowa State University Professional Advancement Grant (J.A.Z.), and National Science Foundation BES 9504383 (D.W.G. and A.T.).

ACKNOWLEDGMENTS

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