Abstract
Labile low-molecular-mass (LMM) transition metal complexes play essential roles in metal ion trafficking, regulation, and signalling in biological systems, yet their chemical identities remain largely unknown due to their rapid ligand-exchange rates and weak M–L bonds. Here, an Escherichia coli cytosol isolation procedure was developed that was devoid of detergents, strongly coordinating buffers, and EDTA. The interaction of the metal ions from these complexes with a SEC column was minimized by pre-loading the column with 67ZnSO4 and then monitoring 66Zn and other metals by inductively coupled plasma mass spectrometry (ICP-MS) when investigating cytosolic ultrafiltration flow-through-solutions (FTSs). Endogenous cytosolic salts suppressed ESI-MS signals, making the detection of metal complexes difficult. FTSs contained ca. 80 µM Fe, 15 µM Ni, 13 µM Zn, 10 µM Cu, and 1.4 µM Mn (after correcting for dilution during cytosol isolation). FTSs exhibited 2–5 Fe, at least 2 Ni, 2–5 Zn, 2–4 Cu, and at least 2 Mn species with apparent masses between 300 and 5000 Da. Fe(ATP), Fe(GSH), and Zn(GSH) standards were passed through the column to assess their presence in FTS. Major LMM sulfur- and phosphorus-containing species were identified. These included reduced and oxidized glutathione, methionine, cysteine, orthophosphate, and common mono- and di-nucleotides such as ATP, ADP, AMP, and NADH. FTSs from cells grown in media supplemented with one of these metal salts exhibited increased peak intensity for the supplemented metal indicating that the size of the labile metal pools in E. coli is sensitive to the concentration of nutrient metals.
Highlights
Transition metals have unique and exceptional catalytic properties which make them indispensable for life [1]
We examined the labile metal content of the cytosol from E. coli and detected numerous LMM labile metal complexes
We initially focussed on zinc because of its redox inactivity and ability to form stable coordination complexes, properties that increased our likelihood of success
Summary
Transition metals have unique and exceptional catalytic properties which make them indispensable for life [1]. We hypothesize that the rate of lability has been adjusted, through evolutionary pressures, to be slow enough for such complexes to “hold together” during transit (to avoid arbitrary deleterious reactions) yet fast enough to release the metal efficiently to its client apoprotein Such trafficking complexes are presumed to have nonproteinaceous ligands composed of metabolites possessing O, N, and/or S Lewis-basic donor atoms. We have attempted to identify labile metal complexes in the cytosol of Escherichia coli but have encountered problems along the way These include unwanted effects of a common chelator, unwanted secondary interactions of labile metals on the column, unwanted ligand-exchange reactions, and the unwanted suppression of ESI-MS signals due to salts present in the cytosol. We have not established the chemical identity or cellular function of these complexes, we are closer to doing so than ever before
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