Analysis of Uncomplexed and Copper-complexed Methanobactin with UV/Visible Spectrophotometry, Mass Spectrometry and NMR Spectrometry

The titration of methanobactin with Cu(II) was monitored by both UV/Vis and NMR spectroscopy and indicates that the major transitions occurring when Cu(II) interacts with methanobactin involve a two-step process that is complete by about 0.6 Cu:mb. This suggests that a 2:1 complex is formed between methanobactin and copper at low copper levels. If this complex exists, it would be different than the 1:1 complex whose structure was solved for in the crystal state. Also, mass spectral data obtained at low copper levels has revealed only 1:1 complexes of methanobactin and copper. NMR and mass spectrometry reveal that some lots of methanobactin contain a second species of methanobactin that is missing its Cterminal methionine residue. Both these species bind Cu(II) and can be readily separated from one another in the copper-complexed state using HPLC. However, attempts to separated the two species in an uncomplexed state leads to extensive degradation of the methanbactin. Evidences suggests that may be due to the presence of dilute acetic acid in the HPLC solvents. Nearly all of the 1H resonances in methanobactin have been assigned and these have produced a couple of surprises. In the copper-complexed state, the amide proton for Tyr5 shows no coupling to its Hα proton. Also, the assignments indicate that the Nterminus in not capped by an isopropyl ester to the THI1 residue, but, more likely is an isobutyl ester. However, the mass spectral data contradicts this conclusion. The Tyr5 HN also produces some other contradictory results, some which suggest a restricted environment when in the the copper-complexed state, and others suggesting a more mobile environment. Up to 90% of the copper can be removed from copper-complexed methanobactin by dialysis against Na2EDTA. NMR spectroscopy shows a structure for the EDTA treated methanobactin that is very similar to that for the copper-complexed methanobactin, indicating that the structural transitions that occur upon copper binding are irreversible.