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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 O2 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 O2-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 O2-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 14N hyperfine couplings attributed to one conformer. The largest, A(14N) = [11.5,11.5,16.0] ± 1.5 MHz, characterizes the unusual bond between one Fe (Fe4) and the backbone amide-N of cysteine-20. The second, A(14N) = [2.8,4.6,3.5] ± 0.3 MHz, is assigned to NC19. The 14N hyperfine couplings are conclusive evidence that Fe4 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 O2 attack. [ABSTRACT FROM AUTHOR]

Catalytic long-range proton transfer in [NiFe]-hydrogenases has long been associated with a highly conserved glutamate (E) situated within 4 Å of the active site. Substituting for glutamine (Q) in the O 2 -tolerant [NiFe]-hydrogenase-1 from Escherichia coli produces a variant (E28Q) with unique properties that have been investigated using protein film electrochemistry, protein film infrared electrochemistry, and X-ray crystallography. At pH 7 and moderate potential, E28Q displays approximately 1% of the activity of the native enzyme, high enough to allow detailed infrared measurements under steady-state conditions. Atomic-level crystal structures reveal partial displacement of the amide side chain by a hydroxide ion, the occupancy of which increases with pH or under oxidizing conditions supporting formation of the superoxidized state of the unusual proximal [4Fe-3S] cluster located nearby. Under these special conditions, the essential exit pathway for at least one of the H + ions produced by H 2 oxidation, and assumed to be blocked in the E28Q variant, is partially repaired. During steady-state H 2 oxidation at neutral pH (i.e., when the barrier to H + exit via Q28 is almost totally closed), the catalytic cycle is dominated by the reduced states "Ni a -R" and "Ni a -C", even under highly oxidizing conditions. Hence, E28 is not involved in the initial activation/deprotonation of H 2 , but facilitates H + exit later in the catalytic cycle to regenerate the initial oxidized active state, assumed to be Ni a -SI. Accordingly, the oxidized inactive resting state, "Ni-B", is not produced by E28Q in the presence of H 2 at high potential because Ni a -SI (the precursor for Ni-B) cannot accumulate. The results have important implications for understanding the catalytic mechanism of [NiFe]-hydrogenases and the control of long-range proton-coupled electron transfer in hydrogenases and other enzymes.