Metal binding and oligomerization properties of FurC (PerR) from Anabaena sp. PCC7120: an additional layer of regulation?

Abstract

Metal and redox homeostasis in cyanobacteria is tightly controlled to preserve the photosynthetic machinery from mismetallation and minimize cell damage. This control is mainly taken by FUR (ferric uptake regulation) proteins. FurC works as the PerR (peroxide response) paralog in Anabaena sp. PCC7120. Despite its importance, this regulator remained poorly characterized. Although FurC lacks the typical CXXC motifs present in FUR proteins, it contains a tightly bound zinc per subunit. FurC:Zn stoichiometrically binds zinc and manganese in a second site, being manganese more efficient in the binding of FurC:Zn to its DNA target PprxA. Oligomerization analyses of FurC:Zn evidence the occurrence of different aggregates ranging from dimers to octamers. Notably, intermolecular disulfide bonds are not involved in FurC:Zn dimerization, being the dimer the most reduced form of the protein. Oligomerization of dimers occurs upon oxidation of thiols by H2O2 or diamide and can be reversed by DTT. Irreversible inactivation of the regulator occurs by metal catalyzed oxidation promoted by ferrous iron. However, inactivation upon oxidation with H2O2 in the absence of iron was reverted by addition of DTT. Comparison of models for FurC:Zn dimers and tetramers obtained using AlphaFold Colab and SWISS-MODEL allowed to infer the residues forming both metal-binding sites and to propose the involvement of Cys86 in reversible tetramer formation. Our results decipher the existence of two levels of inactivation of FurC:Zn of Anabaena sp. PCC7120, a reversible one through disulfide-formed FurC:Zn tetramers and the irreversible metal catalyzed oxidation. This additional reversible regulation may be specific of cyanobacteria.

FurC (PerR) contributes to the regulation of peptidoglycan remodeling and intercellular molecular transfer in the cyanobacterium Anabaena sp. strain PCC 7120

“…The composition of the extracellular milieu defines microbial interactions with the environment-modulating processes such as intercellular communication, toxin production, or biofilm formation, tailoring bacterial ability to cope with different stresses ( 28 , 48 , 49 ). Previous studies point to FurC as an essential stress regulator, playing a central function in the modulation of peroxide, as well as light and nitrogen responses in Anabaena ( 30 , 31 , 50 – 52 ). Furthermore, genome-wide identification of novel direct FurC targets unveiled the role of this protein in the regulation of central carbon metabolism ( 53 ).…”

Expanding the FurC (PerR) regulon in Anabaena (Nostoc) sp. PCC 7120: Genome-wide identification of novel direct targets uncovers FurC participation in central carbon metabolism regulation

“…This fact is very interesting since the MCO mechanism is a complex mechanism that depends on the oxidative stress found in the cell but also on the iron and manganese availability so that FurC could be regulating its target genes integrating these three different signals. In fact, this mechanism of regulation in response to oxidative stress of FurC might be even more intricated, since a new in vitro regulatory mechanism for FurC activity was recently reported, evidencing that apart from the MCO, FurC undergoes a reversible oxidation through interdimer disulfide bridges formation under mild oxidant conditions and iron deficiency that reversibly prevents the in vitro binding to the DNA [ 5 ].…”

“…PCC 7120 cells allowing the derepression of antioxidant enzymes to face oxidative stress situations [ 4 ]. More recently, it was shown that in fact the irreversible inactivation of FurC/PerR by Fe 2+ in aerobic conditions goes along with the incorporation of an oxygen atom into its structure as its observed for other PerR orthologs [ 5 , 6 ]. This study also disclosed the FurC/PerR metal binding and oligomerization properties describing an additional layer of regulation of the protein based on cysteine-based reversible oxidation, increasing the complexity of its regulatory mechanism.…”

Manganese‐dependent transcription regulation by MntR and PerR in Thermus thermophilusHB8

“…All REPA experiments were performed under aerobic conditions and in the presence of a reducing agent, which can help catalyze hydroxyl radical formation in the presence of iron (Kim et al, 1985;Netto & Stadtman, 1996). Similar reaction conditions have been shown to induce metal-catalyzed histidine oxidation of PerR regulators (Sarasa-Buisan et al, 2022). To determine whether TtPerR is susceptible to histidine oxidation in vitro, we incubated TtPerR aerobically in the presence of a reducing agent, supplemented with either buffer or Fe 2+ , and then analyzed protein composition by MALDI-TOF mass spectrometry.…”