Methanobactin (mb) is a small copper-binding peptide produced by methanotrophic bacteria and is intimately involved in both their copper metabolism and their role in the global carbon cycle. The structure for methanobactin comprises seven amino acids plus two chromophoric residues that appear unique to methanobactin. In a previously published structure, both chromophoric residues contain a thiocarbonyl attached to a hydroxyimidazolate ring. In addition, one is attached to a pyrrolidine ring, while the other to an isopropyl ester. A published X-ray determined structure for methanobactin shows these two chromophoric groups forming an N2S2 binding site for a single Cu(I) ion with distorted tetrahedral geometry. In this report we show that NMR, mass spectrometry, and chemical data, reveal a chemical structure that is significantly different than the previously published one. Specifically, the 1H and 13C NMR assignments are inconsistent with an N-terminal isopropyl ester and point instead to a 3-methylbutanoyl group. Our data also indicate that oxazolone rings instead of hydroxyimidazolate rings form the core of the two chromophoric residues. Because these rings are directly involved in the binding of Cu(I) and other metals by methanobactin, and are likely involved in the many chemical activities displayed by methanobactin, their correct identity is central to developing an accurate and detailed understanding of methanobactin’s many chemical and biological roles. For example, the oxazolone rings make methanobactin structurally more similar to other bacterially produced bactins and siderophores and suggest pathways for its biosynthesis.

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Methanobactin (mb) is a chalkophore produced by Methylosinus trichosporium OB3b. This chromopeptide appears to be a part of the copper acquisition system of these methane‐oxidizing bacteria. We have obtained detailed NMR spectra of mb after titration with Cu(II). This is only possible because mb reduces Cu(II) to Cu(I) upon binding and thus becomes diamagnetic as well as slightly less soluble. Cu(I) in solution is typically unstable; however, it remains stable in solution when bound to mb. We have not yet identified the specific reductant which is also capable of reducing Hg(II), Ag(I), and Au(III). Mb is thought to bind metal ions using nitrogen, sulfur and possibly oxygen ligands from its 4‐thiocarbonyl‐5‐hydroxy imidazole (THI), 4‐hydroxy‐5‐thiocarbonyl imidazole (HTI), and possibly its tyrosine. Our experiments show that there are considerable changes in these residue environments denoted by significant changes in the proton NMR spectra of mb when it is bound to copper versus copper‐free mb. Residue assignments have been made using COSY and TOCSY measurements. Protons on the nitrogens of HTI and THI have been identified using HSQC 15N NMR.