Reversible cycling between cysteine persulfide-ligated [2Fe-2S] and cysteine-ligated [4Fe-4S] clusters in the FNR regulatory protein.

Fumarate and nitrate reduction (FNR) regulatory proteins are O(2)-sensing bacterial transcription factors that control the switch between aerobic and anaerobic metabolism. Under anaerobic conditions 4Fe-4S-FNR exists as a DNA-binding homodimer. In response to elevated oxygen levels, the 4Fe-4S cluster undergoes a rapid conversion to a 2Fe-2S cluster, resulting in a dimer-to-monomer transition and loss of site-specific DNA binding. In this work, resonance Raman and UV-visible absorption/CD spectroscopies and MS were used to characterize the interconversion between 4Fe-4S and 2Fe-2S clusters in Escherichia coli FNR. Selective (34)S labeling of the bridging sulfides in the 4Fe-4S cluster-bound form of FNR facilitated identification of resonantly enhanced Cys(32)S-(34)S stretching modes in the resonance Raman spectrum of the O(2)-exposed 2Fe-2S cluster-bound form of FNR. This result indicates O(2)-induced oxidation and retention of bridging sulfides in the form of 2Fe-2S cluster-bound cysteine persulfides. MS also demonstrates that multiple cysteine persulfides are formed on O(2) exposure of 4Fe-4S-FNR. The 4Fe-4S cluster in FNR can also be regenerated from the cysteine persulfide-coordinated 2Fe-2S cluster by anaerobic incubation with DTT and Fe(2+) ion in the absence of exogenous sulfide. Resonance Raman data indicate that this type of cluster conversion involving sulfide oxidation is not unique to FNR, because it also occurs in O(2)-exposed forms of O(2)-sensitive [4Fe-4S] clusters in radical S-adenosylmethionine enzymes. The results provide fresh insight into the molecular mechanism of O(2) sensing by FNR and iron-sulfur cluster conversion reactions in general, and suggest unique mechanisms for the assembly or repair of biological [4Fe-4S] clusters.