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Oxygen-Tolerant [NiFe]-Hydrogenases: The Individual and Collective Importance of Supernumerary Cysteines at the Proximal Fe-S Cluster

2011; American Chemical Society; Volume: 133; Issue: 42 Linguagem: Inglês

10.1021/ja205393w

1943-2984

Michael J. Lukey, Maxie M. Roessler, Alison Parkin, Rhiannon M. Evans, Rosalind A. Davies, Oliver Lenz, Bäerbel Friedrich, Frank Sargent, Fräser A. Armstrong,

Advanced battery technologies research

An important clue to the mechanism for O2 tolerance of certain [NiFe]-hydrogenases is the conserved presence of a modified environment around the iron–sulfur cluster that is proximal to the active site. The O2-tolerant enzymes contain two cysteines, located at opposite ends of this cluster, which are glycines in their O2-sensitive counterparts. The strong correlation highlights special importance for electron-transfer activity in the protection mechanism used to combat O2. Site-directed mutagenesis has been carried out on Escherichia coli hydrogenase-1 to substitute these cysteines (C19 and C120) individually and collectively for glycines, and the effects of each replacement have been determined using protein film electrochemistry and electron paramagnetic resonance (EPR) spectroscopy. The "split" iron–sulfur cluster EPR signal thus far observed when oxygen-tolerant [NiFe]-hydrogenases are subjected to oxidizing potentials is found not to provide any simple, reliable correlation with oxygen tolerance. Oxygen tolerance is largely conferred by a single cysteine (C19), replacement of which by glycine removes the ability to function even in 1% O2.