A Two-α-Helix Extra Domain Mediates the Halophilic Character of a Plant-Type Ferredoxin from Halophilic Archaea,

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Abstract
The [2Fe-2S] ferredoxin (HsFdx) of the halophilic archaeon Halobacterium salinarum exhibits a high degree of sequence conservation with plant-type ferredoxins except for an insertion of 30 amino acids near its N-terminus which is extremely rich in acidic amino acids. Unfolding studies reveal that HsFdx has an unfolding temperature of ∼85 °C in 4.3 M NaCl, but of only 50 °C in low salinity, revealing its halophilic character. The three-dimensional structure of HsFdx was determined by NMR spectroscopy, resulting in a backbone rmsd of 0.6 Å for the diamagnetic regions of the protein. Whereas the overall structure of HsFdx is very similar to that of the plant-type ferredoxins, two additional α-helices are found in the acidic extra domain. 15N NMR relaxation studies indicate that HsFdx is rigid, and the flexibility of residues is similar throughout the molecule. Monitoring protein denaturation by NMR did not reveal differences between the core fold and the acidic domain, suggesting a cooperative unfolding of both parts of the molecule. A mutant of the HsFdx in which the acidic domain is replaced with a short loop of the nonhalophilic Anabaena ferredoxin shows a considerably changed expression pattern. The halophilic wild-type protein is readily expressed in large amounts in H. salinarum, but not in Escherichia coli, whereas the mutant ferredoxin could only be overexpressed in E. coli. The salt concentration was also found to play a critical role for the efficiency of cluster reconstitution: the cluster of HsFdx could be reconstituted only in a solution containing molar concentrations of NaCl, while the reconstitution of the cluster in the mutant protein proceeds efficiently in low salt. These findings suggest that the acidic domain mediates the halophilic character which is reflected in its thermostability, the exclusive expression in H. salinarum, and the ability to efficiently reconstitute the iron−sulfur cluster only at high salt concentrations.