Copper complexation of methanobactin isolated from Methylosinus trichosporium OB3b: pH-dependent speciation and modeling.

Methanobactins are copper-binding ligands produced by aerobic methanotrophic microorganisms. A quantitative understanding of their potential role in methanotrophic copper acquisition requires the investigation of their copper complexes under relevant pH conditions. In this study, a chemical speciation model describing the pH-dependence of copper binding and the formation of the different complexes by methanobactin (mb) is released by Methylosinus trichosporium OB3b was developed. Potentiometric and spectrophotometric titrations of the free ligand indicated the presence of four protonation sites consistent with the molecular structure of methanobactin. Metal titrations revealed a distinct pH-dependence of copper binding to methanobactin between pH 5 and 8. Based on evidence from size-exclusion chromatography coupled to inductively coupled plasma mass spectrometry (ICP-MS), the copper binding was quantitatively described with three different types of copper-methanobactin complexes which can additionally undergo protonation reactions. The high affinity observed upon initial copper additions resulted from the predominant occurrence of copper-methanobactin dimer complexes, mb(2)H(4)Cu and mb(2)H(3)Cu with log K values of 58 and 52, respectively. With increasing copper to methanobactin ratios, methanobactin bound copper as monomers, mbHCu (log K=25) and mbCu (log K=18), whereas at elevated copper activities methanobactin was able to bind two copper ions (mbHCu(2) and mbCu(2)). Model calculations based on the fitted complexation constants suggest that in natural systems, copper-methanobactin complexes are mostly present as monomers.

Isolation and purification of Cu-free methanobactin from Methylosinus trichosporium OB3b.

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.

Effects of anionic surfactants on ligand-promoted dissolution of iron and aluminum hydroxides.

We investigated the influence of the surfactants sodium dodecyl sulfate (SDS) and rhamnolipid (RhL) on ligand-promoted dissolution of goethite (alpha-FeOOH) and boehmite (gamma-AlOOH) at pH 6. The siderophore desferrioxamine B (DFOB), its derivate desferrioxamine D (DFOD), ethylenediaminetetraacetic acid (EDTA), and 8-hydroxyquinoline-5-sulfonic acid (HQS) were used as ligands. The rates of ligand-promoted dissolution of goethite were significantly increased in the presence of low concentrations of anionic surfactants (<80 microM SDS; <6 mg/L RhL). At higher surfactant concentrations, however, the effects of surfactants were negligible. The dissolution rates in the presence of surfactants were not correlated with adsorbed amounts of ligands. Three possible factors contributing to these observations were further investigated and discussed: (i) adsorbed surfactants may influence ligand adsorption by changes in the ligand's surface speciation, (ii) re-adsorption of Fe-DFOB or Fe-DFOD complexes may lead to an underestimation of siderophore-promoted dissolution rates at high surfactant concentrations, and (iii) co-adsorption of protons to goethite with SDS may influence the dissolution rates. However, our results show that none of these three factors can satisfactorily explain the observed effects of anionic surfactants on ligand-promoted dissolution rates of iron and aluminum hydroxides.

Low concentrations of surfactants enhance siderophore-promoted dissolution of goethite.

Surface-active agents (surfactants) are released by many soil bacteria and plant roots and are also important as environmental contaminants. Their presence at interfaces could influence important biogeochemical processes in soils such as ligand-controlled dissolution, an important process in biological iron acquisition. To investigate their potential influence on ligand-controlled dissolution of iron oxides, we studied the dissolution kinetics of goethite (alpha FeOOH) at pH 6 in the presence of the bacterial siderophore desferrioxamine B (DFOB) and the anionic surfactant sodium dodecyl sulfate (SDS). The adsorption isotherm of SDS on goethite showed an increase in the slope at concentrations ranging between 300 and 400 microM SDS in solution. This increase in slope suggested the onset of admicelle formation. Adsorption of DFOB onto goethite increased strongly with increasing concentrations of adsorbed SDS. Small concentrations of SDS (5 microM) resulted in a 3-fold acceleration of DFOB-controlled goethite dissolution in the presence of 80 microM DFOB, compared to the suspensions without SDS. The effects of SDS on the goethite dissolution rates were less pronounced at higher SDS concentrations, and became negligible above 600 microM total SDS. The dissolution rates of goethite were not proportional to the adsorbed DFOB concentrations, as would be expected for ligand-controlled dissolution. We speculate that increasing concentrations of adsorbed SDS result in a change in DFOB surface speciation from inner-sphere to outer-sphere complexes and, consequently, the ligand-controlled dissolution rates are not linearly related to the adsorbed DFOB concentration. Our results provide the first evidence for an important role of biosurfactants in biological iron acquisition involving siderophores.