[NiFe] hydrogenases are metalloenzymes that act as highly efficient molecular electrocatalysts for the interconversion of protons and molecular hydrogen. Unlike any other known molecular electrocatalyst, the members of a subgroup of respiratory membrane-bound [NiFe] hydrogenases are able to maintain H<sub>2</sub> catalysis in the sustained presence of O<sub>2</sub>. This O<sub>2</sub>-tolerance depends on the ability to respond to oxidative inactivation by O<sub>2</sub> by exclusively forming rapidly reactivated active site states, thus implying a catalytic cycle in which O<sub>2</sub> acts as a competing substrate to H<sub>2</sub>. Using isotope ratio mass spectrometry it is proven that the O2-tolerant Escherichia coli Hydrogenase 1 responds to O<sub>2</sub> attack by acting as a four-electron oxidoreductase, catalysing the reaction 2 H<sub>2</sub> + O<sub>2</sub> → 2 H<sub>2</sub>O, equivalent to hydrogen combustion. Special features of the enzyme’s electron relay system enable delivery of the required electrons. A small fraction of the H<sub>2</sub>O produced arises from side reactions proceeding via reactive oxygen species, an unavoidable consequence of the presence of low-potential relay centres that release electrons from H<sub>2</sub> oxidation. While the ability to fully reduce O<sub>2</sub> to harmless H<sub>2</sub>O at the active site to generate the rapidly reactivated state Ni-B, determines if a hydrogenase is O<sub>2</sub>-tolerant, the ratio of oxidative inactivation to reductive reactivation rates determines how tolerant the enzyme is. It is shown by protein film electrochemistry that the (αβ)<sub>2</sub> dimeric assembly of Hyd-1 plays an important role in O<sub>2</sub>-tolerance by aiding reactivation of one catalytic unit through electron transfer from the other. The teamwork between two redundant partners implicates a new role for dimerisation and represents a new example of cooperativity in biology. Finally, the non-natural amino acid p-azido-L-phenylalanine was synthesised and incorporated into Hyd-1, testing the possibility of introducing labels at specific sites.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:640081 |
| Date | January 2014 |
| Creators | Wulff, Philip |
| Contributors | Armstrong, Fraser A. |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://ora.ox.ac.uk/objects/uuid:9e434467-d50b-484a-a17e-ef3091636269 |
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