Divalent metal cations are essential to the structure and function of the ribosome. Previous characterizations of the ribosome performed under standard laboratory conditions have implicated Mg2+ as a primary mediator of ribosomal structure and function. Possible contributions of Fe2+ as a ribosomal cofactor have been largely overlooked, despite the ribosome's early evolution in a high Fe2+ environment, and its continued use by obligate anaerobes inhabiting high Fe2+ niches. Here we show that (i) Fe2+ cleaves RNA by in-line cleavage, a non-oxidative mechanism that has not been detected previously for this metal, (ii) the first-order rate constant with respect to divalent cations is hundreds of times greater with Fe2+ than with Mg2+, (iii) functional ribosomes are associated with Fe2+ after purification from cells grown under low O2 and high Fe2+, and (iv) a small fraction of Fe2+ that is associated with the ribosome is not exchangeable with surrounding divalent cations, presumably because it is tightly coordinated by rRNA and buried in the ribosome. In total, these results expand the ancient role of iron in biochemistry, suggest a novel method for regulation of translation by iron, and highlight a possible new mechanism of iron toxicity.### Competing Interest StatementThe authors have declared no competing interest.

Cutting in-line with iron: ribosomal function and non-oxidative RNA cleavage