Hydrogenases are metalloenzymes that catalyze the reversible oxidation of H 2 . 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 Chlamydomonas reinhardtii (CrHydA1), 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 red H + , H sred H + , 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.

A series of viologen related redox mediators of varying reduction potential has been characterized and their utility as electron shuttles between CdSe quantum dots and hydrogenase enzyme has been demonstrated.

This study reports how the length of capping ligands on a nanocrystal surface affects its interfacial electron transfer (ET) with surrounding molecular electron acceptors, and consequently, impact the H 2 production of a biotic−abiotic hybrid artificial photosynthetic system. Specifically, we study how the H 2 production efficiency of a hybrid system, combining CdS nanorods (NRs), [NiFe] hydrogenase, and redox mediators (propyl-bridged 2,2′-bipyridinium, PDQ 2+ ), depends on the alkyl chain length of mercaptocarboxylate ligands on the NR surface. We observe a minor decrease of the quantum yield for H 2 production from 54 ± 6 to 43 ± 2% when varying the number of methylene units in the ligands from 2 to 7. In contrast, an abrupt decrease of the yield was observed from 43 ± 2 to 4 ± 1% when further increasing n from 7 to 11. ET studies reveal that the intrinsic ET rates from the NRs to the electron acceptor PDQ 2+ are all within 10 8 −10 9 s −1 regardless of the length of the capping ligands. However, the number of adsorbed PDQ 2+ molecules on NR surfaces decreases dramatically when n ≥ 10, with the saturating number changing from 45 ± 5 to 0.3 ± 0.1 for n = 2 and 11, respectively. These results are not consistent with the commonly perceived exponential dependence of ET rates on the ligand length. Instead, they can be explained by the change of the accessibility of NR surfaces to electron acceptors from a disordered "liquid" phase at n < 7 to a more ordered "crystalline" phases at n > ∼7. These results highlight that the order of capping ligands is an important design parameter for further constructing nanocrystal/molecular assemblies in broad nanocrystal-based applications.

Oxidoreductase enzymes often perform technologically useful chemical transformations using abundant metal cofactors with high efficiency under ambient conditions. The understanding of the catalytic mechanism of these enzymes is, however, highly dependent on the availability of well-characterized and optimized time-resolved analytical techniques. We have developed an approach for rapidly injecting electrons into a catalytic system using a photoactivated nanomaterial in combination with a range of redox mediators to produce a potential jump in solution, which then initiates turnover via electron transfer (ET) to the catalyst. The ET events at the nanomaterial-mediator-catalyst interfaces are, however, highly sensitive to the experimental conditions such as photon flux, relative concentrations of system components, and pH. Here, we present a systematic optimization of these experimental parameters for a specific catalytic system, namely, [FeFe] hydrogenase from Chlamydomonas reinhardtii (CrHydA1). The developed strategies can, however, be applied in the study of a wide variety of oxidoreductase enzymes. Our potential jump system consists of CdSe/CdS core–shell nanorods as a photosensitizer and a series of substituted bipyridinium salts as mediators with redox potentials in the range from −550 to −670 mV (vs SHE). With these components, we screened the effect of pH, mediator concentration, protein concentration, photosensitizer concentration, and photon flux on steady-state photoreduction and hydrogen production as well as ET and potential jump efficiency. By manipulating these experimental conditions, we show the potential of simple modifications to improve the tunability of the potential jump for application to study oxidoreductases.

In this study we present the synthesis of Cu2ZnSnSe4 (CZTSe) nanoparticles by microwave-