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Methanobactin, a Copper-Acquisition Compound from Methane-Oxidizing Bacteria

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Science  10 Sep 2004:
Vol. 305, Issue 5690, pp. 1612-1615
DOI: 10.1126/science.1098322

Abstract

Siderophores are extracellular iron-binding compounds that mediate iron transport into many cells. We present evidence of analogous molecules for copper transport from methane-oxidizing bacteria, represented here by a small fluorescent chromopeptide (C45N12O14H62Cu, 1216 daltons) produced by Methylosinus trichosporium OB3b. The crystal structure of this compound, methanobactin, was resolved to 1.15 angstroms. It is composed of a tetrapeptide, a tripeptide, and several unusual moieties, including two 4-thionyl-5-hydroxy-imidazole chromophores that coordinate the copper, a pyrrolidine that confers a bend in the overall chain, and an amino-terminal isopropylester group. The copper coordination environment includes a dual nitrogen- and sulfur-donating system derived from the thionyl imidazolate moieties. Structural elucidation of this molecule has broad implications in terms of organo-copper chemistry, biological methane oxidation, and global carbon cycling.

The mechanisms involved in microbial copper homeostasis are rapidly being elucidated, although the workings of such systems are only understood in model organisms such as Escherichia coli, Enterococcus hirae, and Saccharomyces cerevisiae (14). In these organisms, copper homeostatic systems are geared toward active detoxification as opposed to accumulation and storage. However, in many methanotrophic bacteria (aerobes that oxidize CH4 for carbon and energy and play a major role in the global carbon cycle), copper homeostasis differs because copper requirements can be up to fourfold higher than iron requirements (57). In such methanotrophs, copper plays a central role in metabolism, regulating expression of two methane monooxygenases: a soluble methane monooxygenase (sMMO) and particulate methane monooxygenase (pMMO) (5, 810). Copper also influences the expression of at least two of the four formaldehyde dehydrogenases (1113), the development of internal membranes (5, 8, 14, 15), and the expression of other polypeptides related to copper regulation or transport (5, 1619).

Given the notable role of copper in methanotroph physiology, we postulated that these organisms possess a specialized copper-trafficking mechanism dedicated to transporting higher amounts of copper while protecting cellular components from its toxic effects. Several low–molecular weight copper-containing compounds, previously called copper-binding compounds (CBCs), were implicated in such a mechanism (5, 7, 2022); however, sizes among CBCs varied, and no complete structures could be determined. We now suspect that many of the compounds identified earlier were actually breakdown products of a primary molecule described here, which we identify as methanobactin.

Methanobactin appears to fulfill all the presumed roles of such a copper-trafficking molecule. Methanobactin accumulates to high amounts in the growth media of Methylosinus trichosporium OB3b and Methylococcus capsulatus Bath when grown under copper-limited conditions; however, it is rapidly internalized into the cell when copper is provided. Furthermore, methanobactin stimulates growth in copper-grown M. trichosporium OB3b with an optimal 1:1 copper:methanobactin binding stoichiometry (23), and copper uptake–deficient mutants accumulate methanobactin in their growth medium in the presence of copper (2022). Lastly, methanobactin co-purifies with pMMO at ratios of 8 to 13 methanobactins per pMMO complex, and the removal of methanobactin results in the loss of pMMO activity in cell-free systems (5, 7). These combined results suggest a previously unknown copper acquisition system in M. trichosporium OB3b, mediated by a molecule or molecules that resemble iron siderophores in other bacteria. Furthermore, given the limited understanding of the molecular structure of pMMO and the mechanism of methane oxidation by this enzyme, investigations into the structure and function of methanobactin are of interest.

Here, we report the complete crystal structure of methanobactin excreted into the growth media by M. trichosporium OB3b. Typically, 15 to 20 mg of this yellowish-red compound is isolated per liter of spent medium with 3-day-old copper-limited cultures [optical density at 600 nm (OD600nm) ∼ 0.7], but the yield is heavily dependent on extracellular copper amounts, copper-to-biomass ratios, and culture ages. Purification of the compound involved solid-phase extraction and reversed-phase high-performance liquid chromatography (RP-HPLC) (2325). Mass spectrometry (MS) of the isolated product showed two predominant ions differing in mass by 62 daltons (Fig. 1). The peak with [M – H] at m/z 1153 was assigned to the molecular ion for the deprotonated compound, whereas the most intense peak at m/z 1215 was assigned to the corresponding copper complex [M – 2H + 63Cu+]. Additionally, this signal shows an isotopic distribution characteristic of copper (69.2% 63Cu, 0% 64Cu, and 30.8% 65Cu).

Fig. 1.

Negative-ion MALDI-TOF mass spectrum of extracellular copper compound from M. trichosporium OB3b showing copper complexation. The isotopic distribution of the most dominant peak is that of copper.

X-ray photoelectron spectroscopy (XPS) analysis (25, 26) of the methanobactin-copper complex indicates that the majority of the copper is present as Cu+. Some Cu2+ is noted after extended exposure to air, but this is atypical of physiological conditions and the predominant copper oxidation state in methanobactin is Cu+ (24). Figure 2 presents the XPS binding energy spectra for the copper 2p region for freshly bound copper-methanobactin and CuO, which indicate two low-intensity satellite features (peaks 2 and 3) and a shifted position of the main peaks (1 and 4) relative to CuO, characteristic of Cu+. This observation is consistent with an earlier electron paramagnetic resonance study (7) and is similar to other cell systems with mediated copper transport (27).

Fig. 2.

XPS binding energy spectra for the copper 2p region for methanobactin and for CuO, a reference compound that has copper in the formal oxidation state of Cu2+. The low intensity of the satellite features (2 and 3) and the shifted position of the Cu 2p1/2 and Cu 2p3/2 peaks (1 and 4) in methanobactin indicate that copper is primarily in the Cu+ oxidation state.

The overall structural features of methanobactin, including amino acid composition and sequence, and N- and C-terminus identification were established by a combination of biochemical and mass spectroscopic analyses (2325). The presence of unusual residues was deduced from a significant mass difference (> 358 daltons) between sequence data and MS investigations. Early results suggested that methanobactin was composed of about 10 to 12 residues arranged in a nonlinear motif with a high affinity for copper. Methanobactin was subsequently crystallized (23, 25), and the structure was resolved by direct methods and refined by full-matrix least-squares methods on F2 to 1.15 Å (28).

Crystallographic data (Table 1) indicate that methanobactin is a small chromopeptide that contains one copper ion per molecule, coordinated by a previously unobserved ligand system with a peptide backbone comprising amino acid and non–amino acid residues. The primary sequence of methanobactin is N-2-isopropylester–(4-thionyl-5-hydroxy-imidazole)–Gly1–Ser2–Cys3–Tyr4–pyrrolidine–(4-hydroxy-5-thionyl-imidazole)–Ser5–Cys6–Met7, with an empirical formula of C45N12O14H62Cu (Fig. 3A). Methanobactin is observed as a crystallographic dimer (fig. S1), although the apparent lack of direct interactions between each component suggests that the dimerization in crystals does not persist in solution and is not physiologically relevant.

Fig. 3.

(A) Schematic drawing of methanobactin along with the International Union of Pure and Applied Chemistry atom numbering scheme used in the text. (B) The copper coordination sphere derived from 4-hydroxy-5-thionyl imidazoles. Atom colors: carbon, gray; oxygen, red; nitrogen, blue; and sulfur, yellow. Copper ion is represented by central red sphere. (C) Stereoview of methanobactin surface modeled with the use of solvent-molecule interaction (probe interaction is 1.4 Å).

Table 1.

Crystallographic data and refinement statistics. A total of 1663 parameters were refined against 698 restraints and 8765 data to give wR(F2) = 0.2464 and S = 1.1 (where S is goodness of fit) for weights of w = 1/[σ2(F2) + (0.1600P)2 + 130.00P], where Embedded Image. The final R factor, R(F), was 0.0824 for the 7025 observed, [F > 4σ (F)], data. The largest shift/standard uncertainty was 0.012 in the final refinement cycle. The final difference map had maxima and minima of 0.624 and –0.393 e/Å3, respectively. Parentheses denote the highest resolution shell. Embedded Image and R factor = ∑|Fobs| – |Fcal|/∑|Fobs|, Fobs > 0.

(C44H54CuN12O15S5) · 10.5(H2O)
Formula weight 1404
Molecular weight (C44H54CuN12O15S5) 1217.2
Data collection
Wavelength 0.71073 Å
Reflections collected 66,370
Refinement
Redundancy 7.55
I/σ 18.23 (14.81)
Rsym (%) 8.05 (37.62)
Resolution (Å) 1.15
R factor 0.0832
Completeness 99.8
Goodness of fit on F2 1.1

Overall, methanobactin can be described as having a very compact pyramid-like shape (Fig. 3B) with the metal complexation site being located at the base of the pyramid and not buried. The isopropylester group folds underneath this surface, creating a tail-like projection and a cleft, and appears to obscure the metal site to some extent. The metal coordination environment is composed of dual N- and S-donating systems that are derived from two 4-thionyl-5-hydroxy imidazolate moieties. The bond distances between the donating sulfur atom and its adjacent carbon are 1.68 Å (in thionyl imidazolate A) and 1.67 Å (in thionyl imidazolate B) (Fig. 3C). The sulfur is thus modeled as a thionyl ligand (C=S–Cu) rather than the more commonly found thiolate (C–S–Cu). The C=S distances agree well with previous synthetic N,S-thionyl donor complexes that possess antibacterial properties (29). Furthermore, the thioamide bonds that link each imidazole moiety to a Gly1 and a Ser2 are found in the thiopeptide antibiotics promoinducin and thiostreptone from the Streptomyces species, which is interesting given that methanobactin has also shown to be bacteriocidal for a variety of Gram-positive bacteria (30). This unusual thiopeptide bond is also found in the nickel enzyme methyl–coenzyme reductase from methanogenic archaea that catalyzes methane formation from methyl–coenzyme M and coenzyme B (31, 32).

The Nϵ atom of each imidazole and the S atom of the two thionyl substituents coordinate the copper in a distorted tetrahedron geometry (Fig. 3C). A solvent molecule is not coordinated to the copper in the crystal structure. The Nϵ(8)-Cu-S(13) and Nϵ(19)-Cu-S(24) bond angles (ligand bite angles) of 85.5° and 88.2°, respectively, deviate from the ideal tetrahedral bond angle (109.5°). Both heterocycle rings along with the thionyl substituents are essentially co-planar. The copper atom lies in the plane of thionyl imidazole B but deviates by 0.87 Å from the plane of thionyl imidazole A. The two planes defining the N,S-chromophoric moieties bisect at nearly perpendicular angles. The copper-to-ligand distances are 2.39 and 2.38 Å for Cu-S(13) and Cu-S(24), respectively, and 2.01 and 2.05 Å for Ne(8)-Cu and Ne(19)-Cu (numbers in parentheses designate relative atom numbers), respectively, and indicate strong interactions (Fig. 3C).

The structure of methanobactin as well as growth and physiological data argues for its function as a copper-sequestration compound (2022). The cells appear to excrete methanobactin continuously, and it accumulates in the culture media under copper-deficient conditions. If copper is provided, methanobactin binds the copper and the methanobactin-copper complex is internalized to the cell, possibly to be associated with pMMO (5, 7, 24). Further, its metal-ion shuttling role is suggested by structural similarities to the amino acid–containing pyoverdin class of iron siderophores, which also have antibacterial properties (3336). In fact, the similarities between methanobactin and the pyoverdin siderophores (e.g., azotobactin and pseudobactin produced by Azotobacter spp. and Pseudomonas spp.) led to the renaming of CBC to methanobactin.

If methanobactin is indeed a “coppersiderophore” or a “chalkophore” (after the Greek for copper), a specialized copper-trafficking or defense mechanism probably exists in organisms that produce the compound. However, whether methanobactin acts exclusively as an extracellular coppersequestering agent or has other in vivo functions related to the delivery and insertion of copper ions to copper-containing proteins like pMMO must still be determined. Regardless, the elucidation of the methanobactin structure has major implications in understanding the molecular mechanism of biological methane oxidation and methane cycling in the environment and may also lead to the identification of other copper-trafficking molecules.

Supporting Online Material

www.sciencemag.org/cgi/content/full/305/5690/1612/DC1

Materials and Methods

Figs. S1 and S2

References and Notes

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