Methanogens have a high demand for iron (Fe) and sulfur (S); however, little is known of how they acquire, deploy, and store these elements and how this, in turn, affects their physiology. Methanogens were recently shown to reduce pyrite (FeS
2
) generating aqueous iron-sulfide (FeS
(aq)
) clusters that are likely assimilated as a source of Fe and S. Here, we compare the phenotype of
Methanococcus voltae
when grown with FeS
2
or ferrous iron (Fe(II)) and sulfide (HS
-
). FeS
2
-grown cells are 33% smaller yet have 193% more Fe than Fe(II)/HS
-
-grown cells. Whole cell EPR revealed similar distributions of paramagnetic Fe, although FeS
2
-grown cells showed a broad spectral feature attributed to intracellular thioferrate-like nanoparticles. Differential proteomic analyses showed similar expression of core methanogenesis enzymes, indicating that Fe and S source does not substantively alter the energy metabolism of cells. However, a homolog of the Fe(II) transporter FeoB and its putative transcriptional regulator DtxR were up-expressed in FeS
2
-grown cells, suggesting that cells sense Fe(II) limitation. Two homologs of IssA, a protein putatively involved in coordinating thioferrate nanoparticles, were also up-expressed in FeS
2
-grown cells. We interpret these data to indicate that, in FeS
2
-grown cells, DtxR cannot sense Fe(II) and therefore cannot down-regulate FeoB. We suggest this is due to the transport of Fe(II) complexed with sulfide (FeS
(aq)
) leading to excess Fe that is sequestered by IssA as a thioferrate-like species. This model provides a framework for the design of targeted experiments aimed at further characterizing Fe acquisition and homeostasis in
M. voltae
and other methanogens.
IMPORTANCE
FeS
2
is the most abundant sulfide mineral in the Earth’s crust and is common in environments inhabited by methanogenic archaea. FeS
2
can be reduced by methanogens, yielding aqueous FeS
(aq)
clusters that are thought to be a source of Fe and S. Here, we show that growth of
Methanococcus voltae
on FeS
2
results in smaller cell size and higher Fe content per cell, with Fe likely stored intracellularly as thioferrate-like nanoparticles. Fe(II) transporters and storage proteins were up-regulated in FeS
2
-grown cells. These responses are interpreted to result from cells incorrectly sensing Fe(II) limitation due to assimilation of Fe(II) as FeS
(aq)
. These findings have implications for our understanding of how Fe/S availability influences methanogen physiology and the biogeochemical cycling of these elements.