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-

[FeFe] hydrogenases are highly active catalysts for hydrogen conversion. Their active site has two components: a [4Fe−4S] electron relay covalently attached to the H 2 binding site and a diiron cluster ligated by CO, CN – , and 2-azapropane-1,3-dithiolate (ADT) ligands. Reduction of the [4Fe−4S] site was proposed to be coupled with protonation of one of its cysteine ligands. Here, we used time-resolved infrared (TRIR) spectroscopy on the [FeFe] hydrogenase from Chlamydomonas reinhardtii ( Cr HydA1) containing a propane-1,3-dithiolate (PDT) ligand instead of the native ADT ligand. The PDT modification does not affect the electron transfer step to [4Fe−4S] H but prevents the enzyme from proceeding further through the catalytic cycle. We show that the rate of the first electron transfer step is independent of the pH, supporting a simple electron transfer rather than a proton-coupled event. These results have important implications for our understanding of the catalytic mechanism of [FeFe] hydrogenases and highlight the utility of TRIR.

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Assemblies of inorganic nanocrystals and natural enzymes are promising systems that combine both the unique and tunable photophysical properties of nanocrystals as well as the high catalytic efficiency and selectivity of bioreaction centers. The key challenge in design of these assemblies is to favor the electron/energy transfer (ET) between the abiotic and biotic materials while retarding the undesired charge recombination. The solution to this challenge relies on both rational design of materials with proper energetic and other physical properties, and, equally important if not more so, the surface condition of each component and the configuration of their assemblies. In this study, we investigate a recently developed nanorod and NiFe hydrogenase (H2ase) assemblies which utilize a redox-mediated approach to shuttle the electron transfer between the nanorods and the H2ase enzymes with an impressive H2 production quantum yield up to 77 %.1 We study the impact of alkyl chain lengths of a common type of capping ligand for nanocrystals, mercaptoalkylcarboxylate, on the H2 production quantum yield of the system and elaborate the ligand’s impact on the underlying ET transfer between NRs and the electron acceptors. We observed the existence of an abrupt decrease of the quantum yield for H2 production of the system when increasing the alkyl chain length of the ligands from n= 7 to 10 (35 % and 12 %, respectively), whereas only a minor performance decrease is observed when n is below 7 (35 % and 42 % for n=7 and 2, respectively). These results are further shown in good agreement with the sudden decrease of the yield of the reduced mediator, propyl-bridged 2-2’-bipyridinium (PDQ2+), during the steady-state photoreduction experiments, suggesting that generation efficiency of the redox equivalents control the overall efficiency of the current redox-mediated systems. Further transient spectroscopic measurements revealed that the intrinsic ET transfer rates from the NR to the mediator PDQ are all on the order of 108 s-1 regardless of the length of the capping ligands. Instead, the amount of the average surface attached PDQ2+ molecules decreases dramatically when increasing the length of n above 7, with a saturated surface coverage of σ=40 to σ =0.5 for n=2 and n= 7, respectively. These results cannot be explained by the commonly perceived ligand length dependent ET transfer by tunneling through a barrier: k = exp (- ) on the nanorod surface, with the distance dependent constants, , reported to between 0.3 to 0.8 Å-1.2,3 Instead, these results are rationalized by the accessibility of CdS surface to electron acceptors, probably due to the change of the ligand configuration from the disordered to ordered phases. These results characterize quantitively the efficiency limiting step in the mediator based CdS nanorod/Hydrogenases assemblies, demonstrate impact of configurational arrangements of ligands on NR surface on its ET behavior, and therefore are important for the design of biotic and abiotic systems for various applications. Acknowledgment Wenxing Yang acknowledges the financial support from the Swedish Research Council for an International Postdoc Fellowship. Reference (1) Chica, B.; Wu, C. H.; Liu, Y.; Adams, M. W. W.; Lian, T.; Dyer, R. B. Balancing Electron Transfer Rate and Driving Force for Efficient Photocatalytic Hydrogen Production in CdSe/CdS Nanorod-[NiFe] Hydrogenase Assemblies. Energy Environ. Sci. 2017, 10 (10), 2245–2255. (2) Tagliazucchi, M.; Tice, D. B.; Sweeney, C. M.; Morris-Cohen, A. J.; Weiss, E. A. Ligand-Controlled Rates of Photoinduced Electron Transfer in Hybrid CdSe Nanocrystal/Poly(Viologen) Films. ACS Nano 2011, 5 (12), 9907–9917. (3) Wilker, M. B.; Utterback, J. K.; Greene, S.; Brown, K. A.; Mulder, D. W.; King, P. W.; Dukovic, G. Role of Surface-Capping Ligands in Photoexcited Electron Transfer between CdS Nanorods and [FeFe] Hydrogenase and the Subsequent H 2 Generation. J. Phys. Chem. C 2018, 122 (1), 741–750. Figure 1