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 colt (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 N-14 hyperfine couplings attributed to one conformer. The largest, A(N-14) = [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(N-14) = [2.8,4.6,3.5] +/- 0.3 MHz, is assigned to N-C19. The N-14 hyperfine couplings are conclusive evidence that Fe-4 is a valence-localized Fe3+ in the superoxidized state, whose formation permits an additional electron to be transferred rapidly back to the active site during O-2 attack.