Investigating the Kinetic Competency of HydA1 [FeFe] Hydrogenase Intermediate States via Time-Resolved Infrared Spectroscopy.

Hydrogenases are metalloenzymes that catalyze the reversible oxidation of H. The [FeFe] hydrogenases are generally biased toward proton reduction and have high activities. Several different catalytic mechanisms have been proposed for the [FeFe] enzymes based on the identification of intermediate states in equilibrium and steady state experiments. Here, we examine the kinetic competency of these intermediate states in the [FeFe] hydrogenase from (HydA1), using a laser-induced potential jump and time-resolved IR (TRIR) spectroscopy. A CdSe/CdS dot-in-rod (DIR) nanocrystalline semiconductor is employed as the photosensitizer and a redox mediator efficiently transfers electrons to the enzyme. A pulsed laser induces a potential jump, and TRIR spectroscopy is used to follow the population flux through each intermediate state. The results clearly establish the kinetic competency of all intermediate populations examined: H, H, HH, HH, and H. Additionally, a new short-lived intermediate species with a CO peak at 1896 cm was identified. These results establish a kinetics framework for understanding the catalytic mechanism of [FeFe] hydrogenases.