title: Oxygen-tolerant [NiFe]-hydrogenases: the individual and collective importance of supernumerary cysteines at the proximal Fe-S cluster.
abstract: An important clue to the mechanism for O(2) 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 O(2)-tolerant enzymes contain two cysteines, located at opposite ends of this cluster, which are glycines in their O(2)-sensitive counterparts. The strong correlation highlights special importance for electron-transfer activity in the protection mechanism used to combat O(2). 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% O(2).
authors.affiliation: Department of Chemistry, University of Oxford, South Parks Road, Oxford OX1 3QR, UK.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Lukey
authors.forename: Maxie M
authors.initials: MM
authors.lastname: Roessler
authors.forename: Alison
authors.initials: A
authors.lastname: Parkin
authors.forename: Rhiannon M
authors.initials: RM
authors.lastname: Evans
authors.forename: Rosalind A
authors.initials: RA
authors.lastname: Davies
authors.forename: Oliver
authors.initials: O
authors.lastname: Lenz
authors.forename: Baerbel
authors.initials: B
authors.lastname: Friedrich
authors.forename: Frank
authors.initials: F
authors.lastname: Sargent
authors.forename: Fraser A
authors.initials: FA
authors.lastname: Armstrong
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D003545:Cysteine / Q000737:chemistry* / Q000378:metabolism; D004563:Electrochemistry; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D006864:Hydrogenase / Q000737:chemistry* / Q000378:metabolism; D007501:Iron / Q000737:chemistry*; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010100:Oxygen / Q000737:chemistry* / Q000378:metabolism; D016415:Sequence Alignment; D013455:Sulfur / Q000737:chemistry*
chemical_list: D013455:Sulfur; D007501:Iron; C043296:nickel-iron hydrogenase; D006864:Hydrogenase; D003545:Cysteine; D010100:Oxygen