Reconsidering the czcD (NiCo) Riboswitch as an Iron Riboswitch

[Image: see text] Recent work has proposed a new mechanism of bacterial iron regulation: riboswitches that undergo a conformational change in response to Fe(II). The czcD (NiCo) riboswitch was initially proposed to be specific for Ni(II) and Co(II), but we recently showed via a czcD-based fluorescent sensor that Fe(II) is also a plausible physiological ligand for this riboswitch class. Here, we provide direct evidence that this riboswitch class responds to Fe(II). Isothermal titration calorimetry studies of the native czcD riboswitches from three organisms show no response to Mn(II), a weak response to Zn(II), and similar dissociation constants (∼1 μM) and conformational responses for Fe(II), Co(II), and Ni(II). Only the iron response is in the physiological concentration regime; the riboswitches’ responses to Co(II), Ni(II), and Zn(II) require 10(3)-, 10(5)-, and 10(6)-fold higher “free” metal ion concentrations, respectively, than the typical availability of those metal ions in cells. By contrast, the “Sensei” RNA, recently claimed to be an iron-specific riboswitch, exhibits no response to Fe(II). Our results demonstrate that iron responsiveness is a conserved property of czcD riboswitches and clarify that this is the only family of iron-responsive riboswitch identified to date, setting the stage for characterization of their physiological function.