Identification of a copper-repressible C-type heme protein of Methylococcus capsulatus (Bath). A member of a novel group of the bacterial di-heme cytochrome c peroxidase family of proteins

Department of Molecular Biology, University of Bergen, Norway.
FEBS Journal (Impact Factor: 4). 01/2006; 272(24):6324-35. DOI: 10.1111/j.1742-4658.2005.05020.x
Source: PubMed


Genomic sequencing of the methanotrophic bacterium, Methylococcus capsulatus (Bath), revealed an open reading frame (MCA2590) immediately upstream of the previously described mopE gene (MCA2589). Sequence analyses of the deduced amino acid sequence demonstrated that the MCA2590-encoded protein shared significant, but restricted, sequence similarity to the bacterial di-heme cytochrome c peroxidase (BCCP) family of proteins. Two putative C-type heme-binding motifs were predicted, and confirmed by positive heme staining. Immunospecific recognition and biotinylation of whole cells combined with MS analyses confirmed expression of MCA2590 in M. capsulatus as a protein noncovalently associated with the cellular surface of the bacterium exposed to the cell exterior. Similar to MopE, expression of MCA2590 is regulated by the bioavailability of copper and is most abundant in M. capsulatus cultures grown under low copper conditions, thus indicating an important physiological role under these growth conditions. MCA2590 is distinguished from previously characterized members of the BCCP family by containing a much longer primary sequence that generates an increased distance between the two heme-binding motifs in its primary sequence. Furthermore, the surface localization of MCA2590 is in contrast to the periplasmic location of the reported BCCP members. Based on our experimental and bioinformatical analyses, we suggest that MCA2590 is a member of a novel group of bacterial di-heme cytochrome c peroxidases not previously characterized.

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    • "Whether or not MopE has a direct role in the reduction of the copper ion found in the Cu(I) binding site remains to be elucidated. It is interesting, however, to note that there is an abundance of C-type cytochromes on the surface of M. capsulatus [62], [63], which, with the exception of the dissimilatory metal-reducing bacteria, are not commonly observed in bacteria [64]. Several of these surface-exposed cytochromes showed a fine-tuned copper-regulated expression between 0 to 1.6 µM copper in the growth medium, i.e. in the concentration range where MopE is expressed [2], [16]. "
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    ABSTRACT: Under copper limiting growth conditions the methanotrophic bacterium Methylococcus capsulatus (Bath) secrets essentially only one protein, MopE*, to the medium. MopE* is a copper-binding protein whose structure has been determined by X-ray crystallography. The structure of MopE* revealed a unique high affinity copper binding site consisting of two histidine imidazoles and one kynurenine, the latter an oxidation product of Trp130. In this study, we demonstrate that the copper ion coordinated by this strong binding site is in the Cu(I) state when MopE* is isolated from the growth medium of M. capsulatus. The conclusion is based on X-ray Near Edge Absorption spectroscopy (XANES), and Electron Paramagnetic Resonance (EPR) studies. EPR analyses demonstrated that MopE*, in addition to the strong copper-binding site, also binds Cu(II) at two weaker binding sites. Both Cu(II) binding sites have properties typical of non-blue type II Cu (II) centres, and the strongest of the two Cu(II) sites is characterised by a relative high hyperfine coupling of copper (A(||) =20 mT). Immobilized metal affinity chromatography binding studies suggests that residues in the N-terminal part of MopE* are involved in forming binding site(s) for Cu(II) ions. Our results support the hypothesis that MopE plays an important role in copper uptake, possibly making use of both its high (Cu(I) and low Cu(II) affinity properties.
    PLoS ONE 08/2012; 7(8):e43146. DOI:10.1371/journal.pone.0043146 · 3.23 Impact Factor
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    • "In accordance with the sMMO activity assay, sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analyses of whole cells revealed polypeptides corresponding to subunits of sMMO only in the ~0 mM culture, while subunits of the pMMO were detected in all cultures, but clearly in increasing abundance in cells harvested from copper-added cultures , demonstrating that high copper-to-biomass growth conditions were achieved at these culture conditions (Fig. 1) (Stanley et al., 1983). The surface-associated proteins were collected by ionic extraction with NaCl as previously described (Karlsen et al., 2005a). The incubation of whole cells with NaCl did not disrupt the cells, as verified with microscopy prior to and after the NaCl treatment (not shown). "
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    ABSTRACT: Identification of surface proteins is essential to understand bacterial communication with its environment. Analysis of the surface-associated proteins of Methylococcus capsulatus (Bath) revealed a highly dynamic structure responding closely to the availability of copper in the medium in the range from approximately 0 to 10 microM. Several c-type cytochromes, including three novel multihaem proteins, are present at the cellular surface, a feature that is otherwise a peculiarity of dissimilatory metal-reducing bacteria. At low copper concentrations, the cytochrome c(553o) and the cytochrome c(553o) family protein, encoded by the MCA0421 and MCA0423 genes, respectively, are major constituents of the surfaceome and show a fine-tuned copper-dependent regulation of expression. Two novel members of the cytochrome c(553o) family were identified: MCA0338 was abundant between 5 and 10 microM copper, while MCA2259 was detected only in the surface fraction obtained from approximately 0 microM copper cultures. The presence at the bacterial surface of several c-type cytochromes, generally involved in energy transduction, indicates strongly that redox processes take place at the bacterial surface. Due to the unique role of copper in the biology of M. capsulatus (Bath), it appears that c-type cytochromes have essential functions in copper homeostasis allowing the cells to adapt to varying copper exposure.
    Molecular Microbiology 09/2008; 70(1):15-26. DOI:10.1111/j.1365-2958.2008.06380.x · 4.42 Impact Factor
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    ABSTRACT: The review generalizes the modern data on the taxonomic, structural, and functional diversity of aerobic methanotrophs growing at 25–50°C (Methylococcus capsulatus), 30–62°C (Methylocaldum szegediense, Methylocaldum gracile, and Methylocaldum tepidum), and 50–65°C (Methylothermus thermalis), which belong mainly to the Gammaproteobacteria. The specific features of adaptation of these methanotrophs to the temperature influences are considered on the metabolic and genetic levels. The recent sensational reports on the discovery and primary characterization of thermoacidophilic methanotrophs of the phylum Verrucomicrobia surviving at extreme pH (1–2) and temperature (65°C) values, corresponding to extremely low levels of CH4 and O2 solubility, are analyzed. The possibilities of implementation of the biotechnological potential of thermophilic and thermotolerant methanotrophs are discussed.
    Microbiology 08/2009; 78(4):387-401. DOI:10.1134/S0026261709040018 · 0.64 Impact Factor
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