The splitting of dinitrogen (N2) and reduction to ammonia (NH3) is a kinetically complex and energetically challenging multistep reaction. In the Haber-Bosch process, N2 reduction is accomplished at high temperature and pressure, whereas N2 fixation by the enzyme nitrogenase occurs under ambient conditions using chemical energy from adenosine 5'-triphosphate (ATP) hydrolysis. We show that cadmium sulfide (CdS) nanocrystals can be used to photosensitize the nitrogenase molybdenum-iron (MoFe) protein, where light harvesting replaces ATP hydrolysis to drive the enzymatic reduction of N2 into NH3 The turnover rate was 75 per minute, 63% of the ATP-coupled reaction rate for the nitrogenase complex under optimal conditions. Inhibitors of nitrogenase (i.e., acetylene, carbon monoxide, and dihydrogen) suppressed N2 reduction. The CdS:MoFe protein biohybrids provide a photochemical model for achieving light-driven N2 reduction to NH3.

We have developed complexes of CdS nanorods capped with 3-mercaptopropionic acid (MPA) and Clostridium acetobutylicum [FeFe]-hydrogenase I (CaI) that photocatalyze reduction of H(+) to H(2) at a CaI turnover frequency of 380-900 s(-1) and photon conversion efficiencies of up to 20% under illumination at 405 nm. In this paper, we focus on the compositional and mechanistic aspects of CdS:CaI complexes that control the photochemical conversion of solar energy into H(2). Self-assembly of CdS with CaI was driven by electrostatics, demonstrated as the inhibition of ferredoxin-mediated H(2) evolution by CaI. Production of H(2) by CdS:CaI was observed only under illumination and only in the presence of a sacrificial donor. We explored the effects of the CdS:CaI molar ratio, sacrificial donor concentration, and light intensity on photocatalytic H(2) production, which were interpreted on the basis of contributions to electron transfer, hole transfer, or rate of photon absorption, respectively. Each parameter was found to have pronounced effects on the CdS:CaI photocatalytic activity. Specifically, we found that under 405 nm light at an intensity equivalent to total AM 1.5 solar flux, H(2) production was limited by the rate of photon absorption (~1 ms(-1)) and not by the turnover of CaI. Complexes were capable of H(2) production for up to 4 h with a total turnover number of 10(6) before photocatalytic activity was lost. This loss correlated with inactivation of CaI, resulting from the photo-oxidation of the CdS capping ligand MPA.

We present a study of the self-assembly, charge-transfer kinetics, and catalytic properties of hybrid complexes of CdTe nanocrystals (nc-CdTe) and Clostridium acetobutylicum [FeFe]-hydrogenase I (H(2)ase). Molecular assembly of nc-CdTe and H(2)ase was mediated by electrostatic interactions and resulted in stable, enzymatically active complexes. The assembly kinetics was monitored by nc-CdTe photoluminescence (PL) spectroscopy and exhibited first-order Langmuir adsorption behavior. PL was also used to monitor the transfer of photogenerated electrons from nc-CdTe to H(2)ase. The extent to which the intramolecular electron transfer (ET) contributed to the relaxation of photoexcited nc-CdTe relative to the intrinsic radiative and nonradiative (heat dissipation and surface trapping) recombination pathways was shown by steady-state PL spectroscopy to be a function of the nc-CdTe/H(2)ase molar ratio. When the H(2)ase concentration was lower than the nc-CdTe concentration during assembly, the resulting contribution of ET to PL bleaching was enhanced, which resulted in maximal rates of H(2) photoproduction. Photoproduction of H(2) was also a function of the nc-CdTe PL quantum efficiency (PLQE), with higher-PLQE nanocrystals producing higher levels of H(2), suggesting that photogenerated electrons are transferred to H(2)ase directly from core nanocrystal states rather than from surface-trap states. The duration of H(2) photoproduction was limited by the stability of nc-CdTe under the reactions conditions. A first approach to optimization with ascorbic acid present as a sacrificial donor resulted in photon-to-H(2) efficiencies of 9% under monochromatic light and 1.8% under AM 1.5 white light. In summary, nc-CdTe and H(2)ase spontaneously assemble into complexes that upon illumination transfer photogenerated electrons from core nc-CdTe states to H(2)ase, with low H(2)ase coverages promoting optimal orientations for intramolecular ET and solar H(2) production.

The conformation of thiol-linked oligonucleotides on the surface of Au nanoparticles was controlled by treatment with 6-mercapto-1-hexanol (MCH). MCH displaces noncovalent base adsorption to the surface, changing the oligo conformation on the Au surface. By controlling MCH concentration and reaction time, a change in effective size (D eff) of the Au−DNA conjugates and improved hybridization ability was observed, suggesting a reduced nonspecific adsorption of the oligo to the Au.

This Article describes the electron transfer (ET) kinetics in complexes of CdS nanorods (CdS NRs) and [FeFe]-hydrogenase I from Clostridium acetobutylicum (CaI). In the presence of an electron donor, these complexes produce H2 photochemically with quantum yields of up to 20%. Kinetics of ET from CdS NRs to CaI play a critical role in the overall photochemical reactivity, as the quantum efficiency of ET defines the upper limit on the quantum yield of H2 generation. We investigated the competitiveness of ET with the electron relaxation pathways in CdS NRs by directly measuring the rate and quantum efficiency of ET from photoexcited CdS NRs to CaI using transient absorption spectroscopy. This technique is uniquely suited to decouple CdS→CaI ET from the processes occurring in the enzyme during H2 production. We found that the ET rate constant (k(ET)) and the electron relaxation rate constant in CdS NRs (k(CdS)) were comparable, with values of 10(7) s(-1), resulting in a quantum efficiency of ET of 42% for complexes with the average CaI:CdS NR molar ratio of 1:1. Given the direct competition between the two processes that occur with similar rates, we propose that gains in efficiencies of H2 production could be achieved by increasing k(ET) and/or decreasing k(CdS) through structural modifications of the nanocrystals. When catalytically inactive forms of CaI were used in CdS-CaI complexes, ET behavior was akin to that observed with active CaI, demonstrating that electron injection occurs at a distal iron-sulfur cluster and is followed by transport through a series of accessory iron-sulfur clusters to the active site of CaI. Using insights from this time-resolved spectroscopic study, we discuss the intricate kinetic pathways involved in photochemical H2 generation in CdS-CaI complexes, and we examine how the relationship between the electron injection rate and the other kinetic processes relates to the overall H2 production efficiency.

Hydrogenases catalyze the interconversion of protons and hydrogen according to the reversible reaction: 2H(+) + 2e(-) ⇆ H(2) while using only the earth-abundant metals nickel and/or iron for catalysis. Due to their high activity for proton reduction and the technological significance of the H(+)/H(2) half reaction, it is important to characterize the catalytic activity of [FeFe]-hydrogenases using both biochemical and electrochemical techniques. Following a detailed electrochemical and photoelectrochemical study of an [FeFe]-hydrogenase from Clostridium acetobutylicum (CaHydA), we now report electrochemical and single-molecule imaging studies carried out on a catalytically active hydrogenase preparation. The enzyme CaHydA, a homologue (70% identity) of the [FeFe]-hydrogenase from Clostridium pasteurianum , CpI, was adsorbed to a negatively charged, self-assembled monolayer (SAM) for investigation by electrochemical scanning tunneling microscopy (EC-STM) techniques and macroscopic electrochemical measurements. The EC-STM imaging revealed uniform surface coverage with sufficient stability to undergo repeated scanning with a STM tip as well as other electrochemical investigations. Cyclic voltammetry yielded a characteristic cathodic hydrogen production signal when the potential was scanned sufficiently negative. The direct observation of the single enzyme distribution on the Au-SAM surface coupled with macroscopic electrochemical measurements obtained from the same electrode allowed the evaluation of a turnover frequency (TOF) as a function of potential for single [FeFe]-hydrogenase molecules.

Crystal structures of Escherichia coli dihydrofolate reductase (ecDHFR, EC 1.5.1.3) in binary complexes with folate, 5-deazafolate (5dfol), and 5,10-dideazatetrahydrofolate (ddTHF) have been refined to R-factors of 13.7%, 14.9%, and 14.5%, respectively, all at 1.9 A. All three are isomorphous with a previously reported binary complex of ecDHFR with methotrexate (MTX), in space group P6(1), two molecules per asymmetric unit [Bolin, J. T., Filman, D. J., Matthews, D. A., Hamlin, R. C., & Kraut, J. (1982) J. Biol. Chem. 257, 13650-13662]. A hitherto unobserved water molecule is hydrogen bonded to the pteridine N5 and O4 in both molecules of the asymmetric unit of the folate complex (but not the 5dfol or ddTHF complexes), supporting the hypothesis that N5 protonation of bound substrate, an important step of the DHFR reaction, occurs by way of such a water molecule. There is no indication of a hydrogen bond between N8 of 5dfol and the backbone carbonyl of Ile-5, suggesting that the bacterial enzyme, unlike the human enzyme [Davies, J. F., II, Delcamp, T. J., Prendergast, N. J., Ashford, V. A., Freisheim, J. H., & Kraut, J. (1990) Biochemistry 29, 9467-9479], does not favor protonation at N8. Perhaps this explains why bacterial DHFR is much less effective than vertebrate DHFR in folate reduction. When the ecDHFR.NADPH complex (space group P3221; M. R. Sawaya, in preparation) is superimposed on the folate and 5dfol complexes, the distances from pteridine C6 to nicotinamide C4 were found to be 2.9 and 2.8 A, respectively, in close agreement with the theoretically calculated optimal distance in the transition state for hydride transfer [Wu, Y. D., & Houk, K. N. (1987) J. Am. Chem. Soc. 109, 906-908, 2226-2227]. In contrast to the planar ring system of folate or 5dfol, the reduced pteridine ring of ddTHF is severely puckered and bent toward the nicotinamide pocket, with the reduced pyridine ring assuming a half-chair type of conformation. This change in shape causes the pteridine ring to bind with O4 closer to Trp-22(N epsilon 1) by over 0.5 A, so that an invariant water molecule now bridges these two atoms with ideal hydrogen bonds. Furthermore, while the pABA rings of folate and 5dfol are nearly coincident and closer to the alpha C helix than to the alpha B helix, those of MTX and ddTHF are displaced along the binding crevice by approximately 1.1 and 0.6 A, respectively, and are equidistant from alpha B and alpha C.(ABSTRACT TRUNCATED AT 400 WORDS)

Complexes of CdS nanorods and [FeFe] hydrogenase I from Clostridium acetobutylicum have been shown to photochemically produce H 2 . This study examines the role of the ligands that passivate the nanocrystal surfaces in the electron transfer from photoexcited CdS to hydrogenase and the H 2 generation that follows. We functionalized CdS nanorods with a series of mercaptocarboxylate surface-capping ligands of varying lengths and measured their photoexcited electron relaxation by transient absorption (TA) spectroscopy before and after hydrogenase adsorption. Rate constants for electron transfer from the nanocrystals to the enzyme, extracted by modeling of TA kinetics, decrease exponentially with ligand length, suggesting that the ligand layer acts as a barrier to charge transfer and controls the degree of electronic coupling. Relative light-driven H 2 production efficiencies follow the relative quantum efficiencies of electron transfer, revealing the critical role of surface-capping ligands in determining the photochemical activity of these nanocrystal−enzyme complexes. Our results suggest that the H 2 production in this system could be maximized with a choice of a surface-capping ligand that decreases the distance between the nanocrystal surface and the electron injection site of the enzyme.