Heme-responsive DNA Binding by the Global Iron Regulator Irr from Rhizobium leguminosarum

Abstract

Heme, a physiologically crucial form of iron, is a cofactor for a very wide range of proteins and enzymes. These include DNA regulatory proteins in which heme is a sensor to which an analyte molecule binds, effecting a change in the DNA binding affinity of the regulator. Given that heme, and more generally iron, must be carefully regulated, it is surprising that there are no examples yet in bacteria in which heme itself is sensed directly by a reversibly binding DNA regulatory protein. Here we show that the Rhizobium leguminosarum global iron regulatory protein Irr, which has many homologues within the ␣-proteobacteria and is a member of the Fur superfamily, binds heme, resulting in a dramatic decrease in affinity between the protein and its cognate, regulatory DNA operator sequence. Spectroscopic studies of wild-type and mutant Irr showed that the principal (but not only) heme-binding site is at a conserved HXH motif, whose substitution led to loss of DNA binding in vitro and of regulatory function in vivo. The R. leguminosarum Irr behaves very differently to the Irr of Bradyrhizobium japonicum, which is rapidly degraded in vivo by an unknown mechanism in conditions of elevated iron or heme, but whose DNA binding affinity in vitro does not respond to heme.

Antiparallel and Interlinked Control of Cellular Iron Levels by the Irr and RirA Regulators of Agrobacterium tumefaciens

“…The N-terminal heme regulatory motif of the B. japonicum Irr protein is absent in many irr homologues (61). In addition, it has been shown in R. leguminosarum that while Irr appears to be a heme binding protein, it differs from the B. japonicum homologue in that it is stable in the presence of heme (60). Finally, it has been shown that the HHH motif in the Irr protein from R. leguminosarum is necessary for binding to DNA, while the role of the HHH motif from B. japonicum remains untested (60).…”

“…Mutation of the HHH motif results in a stable Irr protein, while mutation of the HRM reduces the rate of degradation (73). The general applicability of this model to other Irr proteins has recently been called into question (60). The N-terminal heme regulatory motif of the B. japonicum Irr protein is absent in many irr homologues (61).…”

“…In addition, it has been shown in R. leguminosarum that while Irr appears to be a heme binding protein, it differs from the B. japonicum homologue in that it is stable in the presence of heme (60). Finally, it has been shown that the HHH motif in the Irr protein from R. leguminosarum is necessary for binding to DNA, while the role of the HHH motif from B. japonicum remains untested (60). In A. tumefaciens, site-directed mutants of Irr that modify its C-terminal HHH motif to either an AAA motif or an AHA motif eliminate this protein's ability to complement either the growth defect or the PPIX overproduction phenotype of the ⌬irr mutant.…”

The Iron-Responsive Regulator Irr Is Required for Wild-Type Expression of the Gene Encoding the Heme Transporter BhuA in Brucella abortus 2308

“…The studies described by Martínez et al (10) suggest that a similar relationship between cellular iron levels and Irr stability may also exist in B. abortus 2308. However, recent studies with another alphaproteobacterium, Rhizobium leguminosarum, suggest that Irr activity in this bacterium is modulated by heme, and Irr is inactivated, but not degraded, when cellular iron levels increase (26). Instead, heme binding to the R. leguminosarum Irr inhibits its DNA binding capacity.…”

“…Instead, heme binding to the R. leguminosarum Irr inhibits its DNA binding capacity. Distinguishing between these two models for regulating Irr function is important because the Brucella Irr, like the R. leguminsarum Irr, does not possess the heme regulatory element (GCPWAD) in its N terminus that is required for the iron-responsive degradation of Irr in Bradyrhizobium japonicum (19,26). As shown in Fig.…”