Chemical rescue of a site-modified ligand to a [4Fe–4S] cluster in PsaC, a bacterial-like dicluster ferredoxin bound to Photosystem I

Abstract

AbstractChemical rescue of site-modified amino acids using externally supplied organic molecules represents a powerful method to investigate structure–function relationships in proteins. Here we provide definitive evidence that aryl and alkyl thiolates, reagents typically used for in vitro iron–sulfur cluster reconstitutions, serve as rescue ligands to a site-specifically modified [4Fe–4S]1+,2+ cluster in PsaC, a bacterial dicluster ferredoxin-like subunit of Photosystem I. PsaC binds two low-potential [4Fe–4S]1+,2+ clusters termed FA and FB. In the C13G/C33S variant of PsaC, glycine has replaced cysteine at position 13 creating a protein that is missing one of the ligating amino acids to iron–sulfur cluster FB. Using a variety of analytical techniques, including non-heme iron and acid-labile sulfur assays, and EPR, resonance Raman, and Mössbauer spectroscopies, we showed that the C13G/C33S variant of PsaC binds two [4Fe–4S]1+,2+ clusters, despite the absence of one of the biological ligands. 19F NMR spectroscopy indicated that the external thiolate replaces cysteine 13 as a substitute ligand to the FB cluster. The finding that site-modified [4Fe–4S]1+,2+ clusters can be chemically rescued with external thiolates opens new opportunities for modulating their properties in proteins. In particular, it provides a mechanism to attach an additional electron transfer cofactor to the protein via a bound, external ligand