EPR spectroscopic studies of the Fe-S clusters in the O2-tolerant [NiFe]-hydrogenase Hyd-1 from Escherichia coli and characterization of the unique [4Fe-3S] cluster by HYSCORE.

The unusual [4Fe-3S] cluster proximal to the active site plays a crucial role in allowing a class of [NiFe]-hydrogenases to function in the presence of O(2) through its unique ability to undergo two rapid, consecutive one-electron transfers. This property helps to neutralize reactive oxygen species. Mechanistic details and the role of the medial and distal clusters remain unresolved. To probe the Fe-S relay, continuous wave and pulse electron paramagnetic resonance (EPR) studies were conducted on the O(2)-tolerant hydrogenase from Escherichia coli (Hyd-1) and three variants with point mutations at the proximal and/or medial clusters. Reduction potentials of the proximal ([4Fe-3S](5+/4+/3+)) and medial ([3Fe-4S](+/0)) clusters were determined by potentiometry. The medial [3Fe-4S](+/0) reduction potential is exceptionally high, implicating a mechanistic role in O(2)-tolerance. Numerous experiments establish that the distal cluster has a ground state S > 1/2 in all three variants and indicate that this is also the case for native Hyd-1. Concurrent with the Hyd-1 crystal structure, EPR data for the 'superoxidized' P242C variant, in which the medial cluster is 'magnetically silenced', reveal two conformations of the proximal [4Fe-3S](5+) cluster, and X-band HYSCORE spectroscopy shows two (14)N hyperfine couplings attributed to one conformer. The largest, A((14)N) = [11.5,11.5,16.0] ± 1.5 MHz, characterizes the unusual bond between one Fe (Fe(4)) and the backbone amide-N of cysteine-20. The second, A((14)N) = [2.8,4.6,3.5] ± 0.3 MHz, is assigned to N(C19). The (14)N hyperfine couplings are conclusive evidence that Fe(4) is a valence-localized Fe(3+) in the superoxidized state, whose formation permits an additional electron to be transferred rapidly back to the active site during O(2) attack.