Mechanism of hydrogen activation by [NiFe] hydrogenases

[NiFe] hydrogenases contain a conserved arginine (R509) that is suspended over the Ni and Fe atoms. Biochemical, crystallographic and electrochemical analysis of an R509K mutant reveal >100-fold lower oxidation activity despite the maintenance of structural integrity. The active site of [NiFe] hydrogenases contains a strictly conserved arginine that suspends a guanidine nitrogen atom 100-fold lower activity than native enzyme. Conversely, the variants D574N, D118A and D118N/D574N, in which the position of the guanidine headgroup is retained, showed 83%, 26% and 20% activity, respectively. The special kinetic requirement for R509 implicates the suspended guanidine group as the general base in H 2 activation by [NiFe] hydrogenases.