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[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 H2 catalysis in the sustained presence of O2. This O2-tolerance depends on the ability to respond to oxidative inactivation by O2 by exclusively forming rapidly reactivated active site states, thus implying a catalytic cycle in which O2 acts as a competing substrate to H2. Using isotope ratio mass spectrometry it is proven that the O2-tolerant Escherichia coli Hydrogenase 1 responds to O2 attack by acting as a four-electron oxidoreductase, catalysing the reaction 2 H2 + O2 → 2 H2O, equivalent to hydrogen combustion. Special features of the enzyme’s electron relay system enable delivery of the required electrons. A small fraction of the H2O 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 H2 oxidation. While the ability to fully reduce O2 to harmless H2O at the active site to generate the rapidly reactivated state Ni-B, determines if a hydrogenase is O2-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)2 dimeric assembly of Hyd-1 plays an important role in O2-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. |
