Aspartate 141 Is the Fourth Ligand of the Oxygen-sensing [4Fe-4S]²⁺ Cluster of Bacillus subtilis Transcriptional Regulator Fnr*

The Bacillus subtilis redox regulator Fnr controls genes of the anaerobic metabolism in response to low oxygen tension. An unusual structure for the oxygen-sensing [4Fe-4S]²⁺ cluster was detected by a combination of genetic experiments with UV-visible and Mössbauer spectroscopy. Asp-141 was identified as the fourth iron-sulfur cluster ligand besides three Cys residues. Exchange of Asp-141 with Ala abolished functional in vivo complementation of an fnr knock-out strain by the mutagenized fnr gene and in vitro DNA binding of the recombinant regulator FnrD141A. In contrast, substitution of Asp-141 with Cys preserved [4Fe-4S]²⁺ structure and regulator function.