Hydrogenases are microbial redox enzymes
that catalyze H2 oxidation and proton reduction (H2 evolution). While
all hydrogenases show high oxidation activities, the majority of
[FeFe]-hydrogenases are excellent H2 evolution catalysts as well. Their
active site cofactor comprises a [4Fe-4S] cluster covalently linked to a
diiron site equipped with carbon monoxide and cyanide ligands that
facilitate catalysis at low overpotential. Distinct proton transfer
pathways connect the active site niche with the solvent, resulting in a
non-trivial dependence of hydrogen turnover and bulk pH. To analyze the
catalytic mechanism of [FeFe]-hydrogenase, we employ in situ infrared
spectroscopy and infrared spectro-electrochemistry. Titrating the pH
under H2 oxidation or H2 evolution conditions reveals the influence of
site-selective protonation on the equilibrium of reduced cofactor
states. Governed by pKa differences across the active site niche and
proton transfer pathways, we find that individual electrons are
stabilized either at the [4Fe-4S] cluster (alkaline pH values) or at the
diiron site (acidic pH values). This observation is discussed in the
context of the natural pH dependence of hydrogen turnover as catalyzed
by [FeFe]-hydrogenase.<br>
Site-selective Protonation of the One-electron Reduced Cofactor in [FeFe]-Hydrogenase
