Isolation and purification of Cu-free methanobactin from Methylosinus trichosporium OB 3 b

Background: The isolation of highly pure copper-free methanobactin is a prerequisite for the investigation of the biogeochemical functions of this chalkophore molecule produced by methane oxidizing bacteria. Here, we report a purification method for methanobactin from Methylosinus trichosporium OB3b cultures based on reversed-phase HPLC fractionation used in combination with a previously reported resin extraction. HPLC eluent fractions of the resin extracted product were collected and characterized with UV-vis, FT-IR, and C-1s NEXAFS spectroscopy, as well as with elemental analysis and ESI-MS. Results: The results showed that numerous compounds other than methanobactin were present in the isolate obtained with resin extraction. Molar C/N ratios, mass spectrometry measurements, and UV-vis spectra indicated that methanobactin was only present in one of the HPLC fractions. On a mass basis, methanobactin carbon contributed only 32% to the total organic carbon isolated with resin extraction. Our spectroscopic results implied that besides methanobactin, the organic compounds in the resin extract comprised breakdown products of methanobactin as well as polysaccharide-like substances. Conclusion: Our results demonstrate that a purification step is indispensable in addition to resin extraction in order to obtain pure methanobactin. The proposed HPLC purification procedure is suitable for semi-preparative work and provides copper-free methanobactin. Introduction Aerobic methanotrophic microorganisms are Gramnegative bacteria that have the unique ability to use methane as their only source of carbon and energy [1]. In a first step of their metabolic pathway, methanotrophs oxidize methane to methanol, a reaction catalyzed by methane monooxygenase (MMO). Whereas most aerobic methanotrophs produce a membranebound, or particulate MMO (pMMO), some strains also produce a cytoplasmic, or soluble MMO (sMMO) under copper-limiting conditions [1,2]. Both types of MMO contain metal atoms which are involved in the electron transfer to the substrate. pMMO contains several copper atoms [3,4] and possibly a diiron center [3], whereas sMMO holds a diiron active site [5,6]. Copper has been found to play a key role in the regulation of pMMO and sMMO expression as well as in the regulation of other metabolic enzymes of methanotrophs [4,5,7,8]. Furthermore, methane oxidation by pMMO critically depends on copper availability [9]. Due to their high copper requirement, methanotrophs necessitate an efficient copper mobilization and uptake system. Studies conducted in the 1990s provided the first evidence for the production of copper binding compounds (CBC) or copper binding ligands (CBL) released by methanotrophic bacteria under copper-depleted conditions [10-13]. Such copper binding ligands are termed chalkophores, in analogy to siderophores involved in the iron uptake in many organisms. Only recently, researchers have succeeded in isolating and elucidating the structure of a chalkophore from the methanotroph Methylosinus trichosporium OB3b, a small chromopeptide showing high affinity for copper that was named methanobactin [14-16]. * Correspondence: iso.christl@env.ethz. ch Institute of Biogeochemistry and Pollutant Dynamics, Department of Environmental Sciences, ETH Zurich, CHN, Universitätstrasse 16, 8092 Zurich, Switzerland Full list of author information is available at the end of the article Pesch et al. Geochemical Transactions 2011, 12:2 http://www.geochemicaltransactions. com/content/12/1/2 © 2011 Pesch et al; licensee Chemistry Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Copper-bound methanobactin isolated from Methylosinus trichosporium OB3b has a molecular mass of 1215 Da and is composed of seven amino acids and two chromophoric residues involved in copper binding [14]. Upon binding to methanobactin, Cu(II) is immediately reduced to Cu(I) [17,18]. Initially, Kim et al. [14] suggested that Cu(I) coordination is associated with two hydroxyimidazolates, each contributing with one N and one S atom to copper binding. Behling et al. [16], however, proposed a revised structure of the oligopeptide methanobactin containing oxazolone rings instead of hydroxyimidazolate rings responsible for copper binding. The structure of the copper-bound methanobactin is shown in Figure 1. The copper binding peptide methanobactin is not only suspected to be involved in copper acquisition from the extracellular phase [10,17], but it may also function as copper chaperone for pMMO [8,10,19], be responsible for the electron flow to pMMO [19-21], and regulate pMMO expression [10,20]. Furthermore, in vitro studies indicated that methanobactin can act as an oxygen radical scavenger [19,20]. Recent studies revealed the influence of methanobactin in natural systems by effectively mobilizing copper from mineral sources and soils [22]. Additionally, methanobactin was found to promote dissolution of copper-doped silicate [23]. Copper-bound methanobactin has been successfully isolated and purified for characterization and structural elucidation by a two-step purification method [14-16,18]. According to the protocols of the two-step purification method, methanobactin is stabilized with copper and isolated by a solid phase extraction followed by a reversed-phase high-performance liquid chromatography (HPLC) purification step. Methanobactin obtained by these procedures is suitable for characterization purposes. Sufficient amounts of copper-free methanobactin, devoid of buffers such as phosphate, are prerequisite to further investigations of the biogeochemical functions of chalkophores. Choi et al. [21] described a one-step purification method to gain copper-free methanobactin. The one-step purification method consists of a resin extraction and has been applied in several studies [22-24]. From the analysis of exudates released by Methylosinus trichosporium OB3b and isolated following the onestep purification protocol published by Choi et al. [21], however, we found that the elemental composition (mainly C, N, and S) diverges largely from the theoretical composition of methanobactin according to the structure proposed by Behling et al. [16]. Here, we report an improved method yielding highly pure, copper-free methanobactin extending the published one-step resin extraction method with a subsequent purification step with HPLC using a reversedphase C18 column. Different fractions were collected from HPLC effluent and analyzed in order to separate pure methanobactin and to characterize other compounds that were present in the product of the onestep resin extraction method. Characterization methods included various spectroscopic techniques, such as UVvis, C-1s near-edge X-ray absorption fine structure (NEXAFS), and Fourier transform infrared (FT-IR) spectroscopy, as well as electrospray ionization mass spectrometry (ESI-MS). Materials and methods