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Title

Original design of an oxygen-tolerant [NiFe] hydrogenase: Major effect of a valine-to-cysteine mutation near the active site

AuthorsLópez de Lacey, Antonio
Issue Date2011
PublisherAmerican Chemical Society
CitationJournal of the American Chemical Society 133: 986- 997 (2011)
AbstractHydrogenases are efficient biological catalysts of H2 oxidation and production. Most of them are inhibited by O2 and a prerequisite for their use in biotechnological applications under air is to improve their oxygen-tolerance. We have previously shown that exchanging the residue at position 74 in the large subunit of the oxygen sensitive [NiFe] hydrogenase from Desulfovibrio fructosovorans could impact the reaction of the enzyme with O2 (S. Dementin et al, J. Am. Chem. Soc. 131 10156-10164 2009; P. P. Liebgott et al., Nat. Chem. Biol. 6 63-70 2010). This residue, a valine in the wild-type enzyme, located at the bottleneck of the gas channel near the active site, has here been exchanged with a cysteine. A thorough characterization using a combination of kinetic, spectroscopic (EPR, FTIR), crystallographic and electrochemical studies demonstrates that the V74C mutant has features of the naturally-occurring oxygen-tolerant membrane-bound hydrogenases (MBH). The mutant is functional during several minutes under O2, has impaired H2-production activity and has a weaker affinity for CO than the WT. Upon exposure to O2, it is converted into the more easily reactivable inactive form, Ni-B, and this inactive state reactivates about 20 times faster than in the WT enzyme. Control experiments carried out with the V74S and V74N mutants support this interpretation indicate that protonation of the position 74 residue is not the reason the mutants reactivate faster then the WT enzyme. The electrochemical behaviour of the V74C mutant towards O2 is intermediate between that of the WT enzyme from Desulfovibrio fructosovorans and the oxygen-tolerant MBH from Aquifex aeolicus.
URIhttp://hdl.handle.net/10261/80666
Identifiersissn: 0002-7863
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