| Summary: | Strong copper binding ligands have been produced by Synechococcus DC2 grown exponentially under copper stressed conditions. Using pseudo-polarography, all cultures were found to produce a strong copper binding ligand with a log K' of 24.7, assuming that copper is in the +1 oxidation state. Weaker ligands were also present but their binding constants were variable. These strong copper binding ligands were extracted into water-methanol solvents from a hydrophobic resin (XAD-16). Pseudo-polarography was used to track the fractions where the strong ligands were recovered. MALDI and ESI were chosen as soft ionisation sources to access the whole molecule. Both ionisation techniques revealed only one complex that had an isotopic signature characteristic of copper (at m/z 697/699) and this complex was present in two of five culture experiments. Further investigation of the 697/699 complex showed that it was present in all eluent fractions containing the electrochemically determined strong copper binding ligand. Tandem mass spectrometry using MALDI revealed several fragments where copper was still bound, with the smallest copper containing ion at m/z 281/283. A loss of 64/66 mass units from two different fragments suggests the loss of CuH which also implies that the complex binds copper in the +1 oxidation state. Aspects of the MS/MS spectra suggest that this could be a peptide bound to copper by thiol containing cysteine groups. However, further work is needed to ascertain the true chemical nature of this compound. This work represents the first investigation to combine electrochemistry with mass spectrometry to reveal the much anticipated chemical nature of strong copper binding ligands, present in the surface ocean. === Science, Faculty of === Chemistry, Department of === Graduate
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