Labile Iron Pool of Isolated Escherichia coli Cytosol Likely Includes Fe-ATP and Fe-Citrate but not Fe-Glutathione or Aqueous Fe.

The existence of labile iron pools (LFePs) in biological systems has been recognized for decades, but their chemical composition remains uncertain. Here, the LFeP in cytosol from Escherichia coli was investigated. Mössbauer spectra of whole vs lysed cells indicated significant degradation of iron-sulfur clusters (ISCs), even using an unusually gentle lysis procedure; this demonstrated the fragility of ISCs. Moreover, the released iron contributed to the non-heme high-spin Fe(II) species in the cell, which likely included the LFeP. Cytosol batches isolated from cells grown with different levels of iron supplementation were passed through a 3 kDa cutoff membrane, and resulting flow-through-solutions (FTSs) were subjected to SEC-ICP-MS. Mössbauer spectroscopy was used to evaluate the oxidation states of standards. FTSs exhibited iron-detected peaks likely due to different forms of Fe-citrate and Fe-nucleotide triphosphate complexes. Fe-Glutathione (GSH) complexes were not detected using physiological concentrations of GSH mixed with either Fe(II) or Fe(III); Fe(II)-GSH was concluded not to be a significant component of the LFeP in E. coli under physiological conditions. Aqueous iron was also not present in significant concentrations in isolated cytosol and is unlikely a major component of the pool. Fe appeared to bind ATP more tightly than citrate, but ATP also hydrolyzed on the timescale of tens of hours. Isolated cytosol contained excess ligands that coordinated the added Fe(II) and Fe(III). The LFeP in healthy metabolically active cells is undoubtedly dominated by the Fe(II) state, but the LFeP is redox-active such that a fraction might be present as stable and soluble Fe(III) complexes especially under oxidatively stressed cellular conditions.

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

Nickel serves critical roles in the metabolism of E. coli and many prokaryotes. Many details of nickel trafficking are unestablished, but a nonproteinaceous low-molecular-mass (LMM) labile nickel pool (LNiP) is thought to be involved. The portion of the cell lysate that flowed through a 3 kDa cutoff membrane, which ought to contain this pool, was analyzed by size-exclusion and hydrophilic interaction chromatographies (SEC and HILIC) with detection by inductively coupled plasma (ICP) and electrospray ionization (ESI) mass spectrometries. Flow-through-solutions (FTSs) contained 11-15 µM Ni, which represented most Ni in the cell. Chromatograms exhibited 4 major Ni-detected peaks. MS analysis of FTS and prepared nickel complex standards established that these peaks arose from Ni(II) coordinated to oxidized glutathione, histidine, aspartate, and ATP. Surprisingly, Ni complexes with reduced glutathione or citrate were not members of the LNiP under the conditions examined. Aqueous Ni(II) ions were absent in the FTS. Detected complexes were stable in chelator-free buffer but were disrupted by treatment with 1,10-phenanthroline or citrate. Titrating FTS with additional NiSO4 suggested that the total nickel-binding capacity of cytosol is approximately 20-45 µM. Members of the LNiP are probably in rapid equilibrium. Previously reported binding constants to various metalloregulators may have overestimated the relevant binding strength in the cell because aqueous metal salts were used in those determinations. The LNiP may serve as both a Ni reservoir and buffer, allowing cells to accommodate a range of Ni concentrations. The composition of the LNiP may change with cellular metabolism and nutrient status.

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

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.

Chromatographic detection of low-molecular-mass metal complexes in the cytosol of Saccharomyces cerevisiae.

Fluorescence-based chelators are commonly used to probe labile low-molecular-mass (LMM) metal pools in the cytosol of eukaryotic cells, but such chelators destroy the complexes of interest during detection. The objective of this study was to use chromatography to directly detect such complexes. Towards this end, 47 batches of cytosol were isolated from fermenting S. cerevisiae yeast cells and passed through a 10 kDa cut-off membrane. The metal contents of the cytosol and resulting flow-through solution (FTS) were determined. FTSs were applied to a size-exclusion LC column located in an anaerobic refrigerated glove box. The LC system was coupled to an online inductively-coupled-plasma mass spectrometer (ICP-MS) for detection of individual metals. Iron-detected chromatograms of cytosolic FTSs from WT cells exhibited 2-4 major species with apparent masses between 500-1300 Da. Increasing the iron concentration in the growth medium 40-fold increased the overall intensity of these peaks. Approximately 3 LMM cytosolic copper complexes with apparent masses between 300-1300 Da were also detected; their LC intensities were weak, but these increased with increasing concentrations of copper in the growth medium. Observed higher-mass copper-detected peaks were tentatively assigned to copper-bound metallothioneins Cup1 and Crs5. FTSs from strains in which Cup1 or the Cox17 copper chaperone were deleted altered the distribution of LMM copper complexes. LMM zinc- and manganese-detected species were also present in cytosol, albeit at low concentrations. Supplementing the growth medium with zinc increased the intensity of the zinc peak assigned to Crs5 but the intensities of LMM zinc complexes were unaffected. Phosphorus-detected chromatograms were dominated by peaks at apparent masses 400-800 Da, with minor peaks at 1000-1500 Da in some batches. Sulfur chromatograms contained a low-intensity peak that comigrated with a glutathione standard; quantification suggested a GSH concentration in the cytosol of ca. 13 mM. A second LMM sulfur peak that migrated at an apparent mass of 100 Da was also evident.