Low-molecular-mass labile metal pools in Escherichia coli: advances using chromatography and mass spectrometry

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.

Protein metalation in biology

“…Associative ligand exchange is proposed for Ni 2+ transfer to InrS but from small molecule complexes such as Ni 2+ histidine that act as metal buffers ( Figure 2 ). Some of the predominant molecules that bind and buffer selected metal ions in the cytosol of different organisms include glutathione [ 35 , 36 ], bacillithiol in Firmicute bacteria [ 37 ], amino acids [ 38 , 39 ] and specialised proteins such as metallothioneins [ 40 ]. The almost nonexistent concentrations of unbound hydrated metals inside living cells thus become irrelevant for metalation with one caveat: these values reflect how tightly the intracellular milieu binds to exchangeable available metals ( Figure 3 a and b).…”

Direct Detection of the Labile Nickel Pool in Escherichia coli: New Perspectives on Labile Metal Pools

“…The pFTS had lower salt concentration and lacked species that were considered unlikely ligands to Ni. Individual standard nickel complexes were prepared by mixing NiSO 4 and stock solutions of individual ligands (previously described in ref ) to a final concentration of 2 μM Ni and various desired, final concentrations of the ligand. Standards were prepared fresh on the day of analysis and stored anaerobically at 5–10 °C prior to injecting them onto columns.…”

“…Standards were diluted 20× in methanol (LC-MS grade, Fisher Chemical) prior to ESI-MS direct injection. Other samples and standards were prepared as described . These standards and samples were analyzed using a Thermo Scientific Q Exactive Focus MS.…”

“…Also, water-exchange rates of aqueous Ni­(II) ions are slower than those of other aqueous divalent transition metal ions and so complex lability was anticipated to be less problematic. Additionally, Ni­(II) ions adsorb less tightly to the SEC column than Zn­(II) or Cu­(II) ions potentially making our results more easily interpretable. Here we report the direct detection and characterization of the LNiP in E.…”

An Appraisal of the Field of Metallomics and the Roles of Metal Ions in Biochemistry and Cell Signaling

“…Only indirectly and by inference do we have knowledge about the biological molecules or metabolites binding these metal ions as ligands in these pools as their very nature of exchanging ligands makes the chemical characterization with a speciation analysis very challenging. In E. coli, sulfur-containing amino acids along with mono-and dinucleotides have been identified as candidate ligands [32]. Glutathione has been suggested to be a ligand for Fe 2+ while multiple ligands have been discussed for Zn 2+ [33,34].…”