Iron and redox sensing in plants: properties of the hemerythrin-like domains of Arabidopsis BRUTUS and BRUTUS-LIKE2 proteins

Iron uptake in plants is negatively regulated by a unique subfamily of ubiquitin E3 ligases, exemplified by Arabidopsis thaliana BRUTUS (BTS). These large proteins are comprised of an usual triple hemerythrin (Hr)-like domain at the N-terminus and a RING-type E3 ligase at the C-terminus. Physiological studies point to the highly conserved BTS proteins as the elusive iron sensors in plants, but biochemical evidence for this has been lacking. Here we characterize the redox properties and iron-binding dynamics of the Hr-like domain in both BTS and BTS-LIKE2 (BTSL2) and propose a model for their atypical mechanism of iron sensing. The recombinantly expressed BTS N-terminal domain contained three diiron centres, one in each Hr-like motif, whereas BTSL2 lacked the second diiron centre, which is positioned at the distal end according to protein models and SAXS data. The diiron centres of BTSL2 were reduced by dithionite and glutathione and could be re-oxidized upon exposure to O ₂ and H ₂ O ₂ . In the reduced state, at least one of the irons of each diiron centre was labile and readily accessible to an Fe ²⁺ chelator. Addition of Fe ²⁺ to the hemi-apoprotein form and subsequent oxidation reconstituted the diiron centres, as monitored by circular dichroism spectroscopy. In the oxidized state, none of the Fe ³⁺ ions were labile. These data show that iron binding to BTS/L is dynamic, in support of their proposed iron-sensing function, but interestingly it is conditional on O ₂ or oxidative stress. We speculate that the dual-sensing mode may be particularly important for photosynthetic organisms, for which labile diiron centres rather than other iron cofactors have been selected during evolution.