Strains lacking and overexpressing
the vacuolar iron (Fe) importer CCC1 were characterized
using Mössbauer and EPR spectroscopies.
Vacuolar Fe import is impeded in Δccc1 cells
and enhanced in CCC1-up cells, causing vacuolar Fe
in these strains to decline and accumulate, respectively, relative
to WT cells. Cytosolic Fe levels should behave oppositely. The Fe
content of Δccc1 cells grown under low-Fe conditions
was similar to that in WT cells. Most Fe was mitochondrial with some
nonheme high spin (NHHS) FeII present. Δccc1 cells grown with increasing Fe concentration in the medium contained
less total Fe, less vacuolar HS FeIII, and more NHHS FeII than in comparable WT cells. As the Fe concentration in
the growth medium increased, the concentration of HS FeIII in Δccc1 cells increased to just 60% of WT
levels, while NHHS FeII increased to twice WT levels, suggesting
that the NHHS FeII was cytosolic. Δccc1 cells suffered more oxidative damage than WT cells, suggesting that
the accumulated NHHS FeII promoted Fenton chemistry. The
Fe concentration in CCC1-up cells was higher than
in WT cells; the extra Fe was present as NHHS FeII and
FeIII and as FeIII oxyhydroxide nanoparticles.
These cells contained less mitochondrial Fe and exhibited less ROS
damage than Δccc1 cells. CCC1-up cells were adenine-deficient on minimal medium; supplementing with
adenine caused a decline of NHHS FeII suggesting that some
of the NHHS FeII that accumulated in these cells was associated
with adenine deficiency rather than the overexpression of CCC1. A mathematical model was developed that simulated
changes in Fe distributions. Simulations suggested that only a modest
proportion of the observed NHHS FeII in both strains was
the cytosolic form of Fe that is sensed by the Fe import regulatory
system. The remainder is probably generated by the reduction of the
vacuolar NHHS FeIII species.