ABSTRACT
Escherichia coli ZinT is a metal binding protein involved in zinc homeostasis, with additional putative functions in the resistance against other metals. Herein, a method was designed and implemented to evaluate from a structural and functional viewpoint metal binding to E. coli ZinT in 96-well microtiter plates. The isolated ZinT was mixed with several metal ions and their binding ability was determined by differential scanning fluorimetry. From the positive hits, six metal ions were evaluated in terms of their toxicity towards an E. coli strain depleted of ZinT (ΔzinT) using as control a strain deleted in the galT gene (ΔgalT). The different sensitivities of each strain to the tested metals revealed novel roles of ZinT in the resistance to cobalt, cadmium and mercury. This approach provides a valuable and reliable platform for the analysis of metal binding and its functional implications, extendable to other metal binding proteins. In combination with the developed platform, structural studies were performed with ZinT, with the zinc-loaded crystallographic structure being obtained at 1.79 Å resolution. Besides the canonical zinc-binding site located near the N-terminus, the herein reported dimeric ZinT structure unravelled extra zinc binding sites that support its role in metal loading and/or transport. Altogether, the designed experimental platform allowed revealing new roles for the ZinT protein in microbial resistance to heavy metal toxicity, as well as structural insights into the ZinT metal binding mechanism.
Subject(s)
Cadmium/toxicity , Cobalt/toxicity , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Mercury/toxicity , Binding Sites , Crystallography, X-Ray , Escherichia coli/drug effects , Escherichia coli Proteins/isolation & purification , Ions , Mutation/genetics , Protein Multimerization/drug effectsABSTRACT
A novel two-component enzyme system from Escherichia coli involving a flavorubredoxin (FlRd) and its reductase was studied in terms of spectroscopic, redox, and biochemical properties of its constituents. FlRd contains one FMN and one rubredoxin (Rd) center per monomer. To assess the role of the Rd domain, FlRd and a truncated form lacking the Rd domain (FlRdDeltaRd), were characterized. FlRd contains 2.9+/-0.5 iron atoms/subunit, whereas FlRdDeltaRd contains 2.1+/-0.6 iron atoms/subunit. While for FlRd one iron atom corresponds to the Rd center, the other two irons, also present in FlRdDeltaRd, are most probably due to a di-iron site. Redox titrations of FlRd using EPR and visible spectroscopies allowed us to determine that the Rd site has a reduction potential of -140+/-15 mV, whereas the FMN undergoes reduction via a red-semiquinone, at -140+/-15 mV (Fl(ox)/Fl(sq)) and -180+/-15 mV (Fl(sq)/Fl(red)), at pH 7.6. The Rd site has the lowest potential ever reported for a Rd center, which may be correlated with specific amino acid substitutions close to both cysteine clusters. The gene adjacent to that encoding FlRd was found to code for an FAD-containing protein, (flavo)rubredoxin reductase (FlRd-reductase), which is capable of mediating electron transfer from NADH to Desulfovibrio gigas Rd as well as to E. coli FlRd. Furthermore, electron donation was found to proceed through the Rd domain of FlRd as the Rd-truncated protein does not react with FlRd-reductase. In vitro, this pathway links NADH oxidation with dioxygen reduction. The possible function of this chain is discussed considering the presence of FlRd homologues in all known genomes of anaerobes and facultative aerobes.
