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.