Cyanide Photodissociation from the Site-Differentiated [4Fe-4S]1+ Cluster of Pyrococcus furiosus Ferredoxin Characterized by EPR and 13C/15N ENDOR

Ferredoxin (Fd) from the hyperthermophilic archaeon Pyrococcus furiosus (Pf), Pf-Fd, functions in electron transfer, serving to transfer an electron from various oxidoreductases to an H2-evolving, membrane-bound hydrogenase. The [4Fe-4S] cluster of Pf-Fd is coordinated by three cysteine residues and the carboxylate group of an aspartate residue. Recently, we have found that several such site-differentiated [4Fe-4S]1+ clusters are photoactive, inspiring us to investigate the photoreactivity of the site-differentiated [4Fe-4S]1+ cluster in Pf-Fd. We show here through X-band EPR and 13C/15N Q-band ENDOR studies that visible-light irradiation of the cyanide-bound [4Fe-4S]1+ cluster of Pf-Fd, Pf-Fd-CN, at 12 K results in photodissociation of the cyanide anion, which geminately recombines only upon annealing to 150 K. In addition, this work revisits our previous CW 13C/15N ENDOR studies of Pf-Fd-CN with pulsed-ENDOR techniques. Of particular note, we definitively determine the absolute sign of the 13C hyperfine coupling with the PESTRE pulsed-ENDOR protocol and from this draw inferences about Fe valence localization within the cyanide-bound cluster.