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HdrC2 from Acidithiobacillus ferrooxidans owns two iron-sulfur binding motifs but binds only one variable cluster between [4Fe-4S] and [3Fe-4S]




Abstract: The heterodisulfide reductase complex HdrABC from Acidithiobacillus ferrooxidans was suggested to own novel features that act in reverse to convert the sulfane sulfur of GSnH species (n>1) into sulfite in sulfur oxidation. The HdrC subunit is potentially encoded by two different highly upregulated genes sharing only 29% identity in A. ferrooxidans grown in sulfur-containing medium, which were named as HdrC1 and HdrC2 respectively and had been confirmed to contain iron-sulfur cluster by expression and characterization, especially the HdrC1 which had been showed to bind only one [4Fe-4S] cluster by mutations. However, the mutations of the HdrC2 remain to be done and the detailed binding information of it is still unclear. Here we report the expression, mutations and molecular modeling of the HdrC2 from A. ferrooxidans. This HdrC2 had two identical motifs (Cx2Cx2Cx3C) containing total of eight cysteine residues potentially for iron-sulfur cluster binding. This purified HdrC2 was exhibited to contain one variable cluster reversibly converted between [4Fe-4S] and [3Fe-4S] according to different conditions by the UV-scanning and EPR spectra. The site-directed mutagenesis results of these eight residues further confirmed that the HdrC2 in reduction with Fe2+ condition loaded only one [4Fe-4S]+ with spin S=1/2 ligated by the residues of Cys73, Cys109, Cys112 and Cys115; the HdrC2 in natural aeration condition lost the Fe atom ligated by the residue of Cys73 and loaded only one [3Fe-4S]0 with spin S=0; the HdrC2 in oxidation condition loaded only one [3Fe-4S]+ with spin S=1/2. Molecular modeling results were well in line with the experiment results and showed that the length of the bond linked between [4Fe-4S] and Cys73 was obviously longer and so looser than that of other three ligated residues respectively, which explained that the Fe atom ligated by Cys73 in [4Fe-4S] was easy to firstly lose and caused the change of the cluster from [4Fe-4S] to [3Fe-4S].

Keywords: Acidithiobacillus ferrooxidans; heterodisulfide reductase; HdrC; expression; mutation; molecular modeling.


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