Biogenesis Of Iron-sulfur Clusters From Acidith Iobacillus Ferrooxidans

Acidithiobacillus ferrooxidans is a widely studied bacterium that lives in acid mine drainage, which obtains energy through the oxidation of ferrous ion to ferric ion with O2as the terminal electron acceptor. Acidithiobacillus ferrooxidans has attracted much attention in studying the mechanism of bioleaching. Iron sulfur clusters are one of the most ancient and ubiquitous redox centre in almost all living organism and play an important role in photosynthesis, respiration and nitrogen fixation. In this study, we usd the Acidithiobacillus ferrooxidans ATCC23270as the type strain to study the biogenesis and transfer mechanism of iron-sulfur clusters. The primary subjects of which include six parts as follows:(1) We cloned the gene of iron-sulfur cluster assembly scaffold protein IscU from the isc operon and constituted the plasmid pET28b-IscU. The recombinant plasmid was successfully expressed in LB medium with0.5mM IPTG under the room temperature overnight. The recombinant IscU protein was expressed as fusion proteins with a His-tag and purified by affinity chromatography using Ni-NTA agarose gel attached with the AKTA explore. IscU had a molecular weight of16kD and are colourless. The purified IscU did not have any iron-sulfur cluster and could not bind iron neither in vitro nor in vivo. IscU colud act as a scaffold protein to assemble the [2Fe-2S] cluster within30min. pH is an important impact factor for biogenesis of iron-sulfur cluster in IscU, the suitable pH value for iron sulfur cluster assemble is pH7.0-8.0, decreasing the pH value will restrain the iron-sulfur cluster assembly.(2) The gene of cysteine desulfurase protein iscS from the isc operon was cloned and constituted the into the plasmid pET28b. The recombinant plasmid pET28b-IscS was successfully expressed in LB medium and purified by affinity chromatography. IscS had a molecular weight of46kD and was light yellow in color. The purified IscS had high cysteine desulfurase activity, could catalyze desulfurization of L-cysteine. Incubating IscS with PLP would greatly increase the activity of IscS. The most suitable condition for IscS cysteine desulfurase activity was37℃and pH8.0. IscS was very important for iron-sulfur cluster assembly both in vitro and in vivo, it could catalyze desulfurization of L-cysteine and transfer sulfur for iron-sulfur cluster assembly on apo-fdx in vitro, inactivation of IscS serious affected the iron-sulfur cluster assembly on fdx in vivo.(3) The gene of IscA from A. ferrooxidans ATCC23270was cloned, expressed and purified by affinity chromatography. The purified IscA had a molecular weight of11kD and was brown in color. Acidithiobacillus ferrooxidans IscA is a [4Fe-4S] cluster binding protein, and it can bind iron in the presence of DTT with an apparent iron association constant of4×1020M-1. The iron binding in IscA can be promoted by oxygen through oxidizing ferrous iron to ferric iron. Furthermore, we show that the iron bound form IscA can be converted to iron sulfur cluster bound form in the presence of IscS and L-cysteine in vitro. Substitution of the invariant cysteine residues Cys35, Cys99or Cys101in IscA abolishes the iron binding activity of the protein; the IscA mutants that fail to bind iron are unable to assemble the iron sulfur clusters. Further studies indicate that the iron-loaded IscA could act as an iron donor for the assembly of iron-sulfur clusters in the scaffold protein IscU in vitro.(4) L-cysteine could mobilize the iron centre in IscA and to provide sulfur via IscS for iron-sulfur cluster assembly in IscU. When equal amounts of IscA and IscU are incubated with ferrous iron in the presence of IscS, L-cysteine and DTT, iron-sulfur cluster are assembled in IscU, suggested IscU is a preferred iron-sulfur cluster assembly scaffold protein. In contract, when equal amounts of IscA and IscU are incubated with ferrous iron in the presence of IscS and DTT, nearly all iorn is bound to IscA, suggested IscA is a preferred iron binding protein. Iron sulfur clusters could be transfer from pre-assembled scaffold proteins to apo-form iron sulfur proteins. Holo-IscA could transfer its [4Fe-4S] cluster to apo-fdx and apo-APS reductase to form [2Fe-2S]-fdx and [4Fe-4S]-APS reductase. After the iron-sulfur cluster transferring, the reconstitution iron sulfur proteins could restore their physiological activities.(5) The gene of ATP sulfurylase cysDl from the cys operon was cloned and constituted the into the plasmid pET28b. The recombinant plasmid pET28b-CysDl was successfully expressed in LB medium and purified by affinity chromatography. CysDl had a molecular weight of33kD and was colorless. The purified ATP sulfurylase could catalysis ATP and sulfate to form APS and pyrophosphate, the specific activity of the purified ATP sulfurylase was3.0×103U/mg. We constructed the mutant expression plasmid (Q170S, R173S, D211S) of the protein using the Site-directed mutagenesis. Molecular modelling for the protein, UV-Vis scanning and enzyme activity revealed that Q170, R173and D211were curcial sites for ATP sulfurylase bindings.