Hydrogen activation by [NiFe]-hydrogenases

Hydrogenase-1 (Hyd-1) from Escherichia coli is a membrane-bound enzyme that catalyses the reversible oxidation of molecular H₂. The active site contains one Fe and one Ni atom and several conserved amino acids including an arginine (Arg⁵⁰⁹), which interacts with two conserved aspartate residues (Asp¹¹⁸ and Asp⁵⁷⁴) forming an outer shell canopy over the metals. There is also a highly conserved glutamate (Glu²⁸) positioned on the opposite side of the active site to the canopy. The mechanism of hydrogen activation has been dissected by site-directed mutagenesis to identify the catalytic base responsible for splitting molecular hydrogen and possible proton transfer pathways to/from the active site. Previous reported attempts to mutate residues in the canopy were unsuccessful, leading to an assumption of a purely structural role. Recent discoveries, however, suggest a catalytic requirement, for example replacing the arginine with lysine (R509K) leaves the structure virtually unchanged, but catalytic activity falls by more than 100-fold. Variants containing amino acid substitutions at either or both, aspartates retain significant activity. We now propose a new mechanism: heterolytic H₂ cleavage is via a mechanism akin to that of a frustrated Lewis pair (FLP), where H₂ is polarized by simultaneous binding to the metal(s) (the acid) and a nitrogen from Arg⁵⁰⁹ (the base).