Investigating the Kinetic Competency of <i>Cr</i>HydA1 [FeFe] Hydrogenase Intermediate States via Time-Resolved Infrared Spectroscopy

Abstract

Hydrogenases are metalloenzymes that catalyze the reversible oxidation of H₂. 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 <i>Chlamydomonas reinhardtii</i> (<i>Cr</i>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_ox, H_red, H_redH⁺, H_sredH⁺, and H_hyd. Additionally, a new short-lived intermediate species with a CO peak at 1896 cm^-1 was identified. These results establish a kinetics framework for understanding the catalytic mechanism of [FeFe] hydrogenases.