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](2+)-FNR exists as a DNA-binding homodimer. In response to elevated oxygen levels, the [4Fe-4S](2+) cluster undergoes a rapid conversion to a [2Fe-2S](2+) 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](2+) and [2Fe-2S](2+) clusters in Escherichia coli FNR. Selective S-34 labeling of the bridging sulfides in the [4Fe-4S](2+) cluster-bound form of FNR facilitated identification of resonantly enhanced Cys(32)S-S-34 stretching modes in the resonance Raman spectrum of the O-2-exposed [2Fe-2S](2+) cluster-bound form of FNR. This result indicates O-2-induced oxidation and retention of bridging sulfides in the form of [2Fe-2S](2+) cluster-bound cysteine persulfides. MS also demonstrates that multiple cysteine persulfides are formed on O-2 exposure of [4Fe-4S](2+)-FNR. The [4Fe-4S](2+) cluster in FNR can also be regenerated from the cysteine persulfide-coordinated [2Fe-2S](2+) cluster by anaerobic incubation with DTT and Fe2+ 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.