Detection and Structural Elucidation of Copper Binding Tri- and Tetrapyrrole Ligands Produced by the Marine Diatom <i>Phaeodactylum Tricornutum</i>

In seawater, most dissolved copper (Cu) is complexed by organic ligands, many of which are thought to be produced by phytoplankton. Although very little is known about the composition and structure of these ligands, they play an important role in determining the reactivity and bioavailability of Cu. In this study, <i>Phaeodactylum tricornutum</i>, a marine diatom known to produce Cu ligands (CuLs), was grown in laboratory pure culture, and the CuLs were recovered from the growth media. Using liquid chromatography coupled to ultrahigh resolution tandem mass spectrometry, 11 Cu ligand complexes were identified and assigned molecular formulas. Molecular formulas were confirmed by comparing the expected and observed relative abundances of ¹⁵N, ¹³C, ⁶⁵Cu, and ¹⁸O isotopologues. The CuLs had molecular weights from 520 to 719 Da and molecular formulas of C_26-35H_23-36O_5-9N_3-4Cu with an average assignment error of 56 ppb. High-resolution tandem mass spectrometry of the Cu-bound and metal-free ligands revealed these to be a suite of tri- and tetrapyrroles stabilized through complexation of Cu by N. The ligands share similar parent structures but differ in the number, type, and arrangement of functional groups that decorate the pyrroles. The similarity of CuL structures with known catabolites of chlorophyll suggests these ligands may be widely produced by marine photoautotrophs.