Proton Transport in <i>Clostridium pasteurianum</i> [FeFe] Hydrogenase I: A Computational Study

To better understand the proton transport through the H₂ production catalysts, the [FeFe] hydrogenases, we have undertaken a modeling and simulation study of the proton transfer processes mediated by amino acid side-chain residues in hydrogenase I from <i>Clostridium pasteurianum</i>. Free-energy calculation studies show that the side chains of two conserved glutamate residues, Glu-279 and Glu-282, each possess two stable conformations with energies that are sensitive to protonation state. Coordinated conformational changes of these residues can form a proton shuttle between the surface Glu-282 and Cys-299, which is the penultimate proton donor to the catalytic H-cluster. Calculated acid dissociation constants are consistent with a proton relay connecting the H-cluster to the bulk solution. The complete proton-transport process from the surface-disposed Glu-282 to Cys-299 is studied using coupled semiempirical quantum-mechanical/classical-mechanical dynamics. Two-dimensional free-energy maps show the mechanisms of proton transport, which involve Glu-279, Ser-319, and a short internal water relay to connect functionally Glu-282 with the H-cluster. The findings of conformational bistability, PT event coupling with p<i>K</i>_a mismatch, and water participation have implications in the design of artificial water reduction or general electrocatalytic H₂-production catalysts

Proton Transport in <i>Clostridium pasteurianum</i> [FeFe] Hydrogenase I: A Computational Study

To better understand the proton transport through the H₂ production catalysts, the [FeFe] hydrogenases, we have undertaken a modeling and simulation study of the proton transfer processes mediated by amino acid side-chain residues in hydrogenase I from <i>Clostridium pasteurianum</i>. Free-energy calculation studies show that the side chains of two conserved glutamate residues, Glu-279 and Glu-282, each possess two stable conformations with energies that are sensitive to protonation state. Coordinated conformational changes of these residues can form a proton shuttle between the surface Glu-282 and Cys-299, which is the penultimate proton donor to the catalytic H-cluster. Calculated acid dissociation constants are consistent with a proton relay connecting the H-cluster to the bulk solution. The complete proton-transport process from the surface-disposed Glu-282 to Cys-299 is studied using coupled semiempirical quantum-mechanical/classical-mechanical dynamics. Two-dimensional free-energy maps show the mechanisms of proton transport, which involve Glu-279, Ser-319, and a short internal water relay to connect functionally Glu-282 with the H-cluster. The findings of conformational bistability, PT event coupling with p<i>K</i>_a mismatch, and water participation have implications in the design of artificial water reduction or general electrocatalytic H₂-production catalysts

Identification of a Catalytic Iron-Hydride at the H‑Cluster of [FeFe]-Hydrogenase

Hydrogenases couple electrochemical potential to the reversible chemical transformation of H₂ and protons, yet the reaction mechanism and composition of intermediates are not fully understood. In this Communication we describe the biophysical properties of a hydride-bound state (H_hyd) of the [FeFe]-hydrogenase from <i>Chlamydomonas reinhardtii</i>. The catalytic H-cluster of [FeFe]-hydrogenase consists of a [4Fe-4S] subcluster ([4Fe-4S]_H) linked by a cysteine thiol to an azadithiolate-bridged 2Fe subcluster ([2Fe]_H) with CO and CN^– ligands. Mössbauer analysis and density functional theory (DFT) calculations show that H_hyd consists of a reduced [4Fe-4S]_H⁺ coupled to a diferrous [2Fe]_H with a terminally bound Fe-hydride. The existence of the Fe-hydride in H_hyd was demonstrated by an unusually low Mössbauer isomer shift of the distal Fe of the [2Fe]_H subcluster. A DFT model of H_hyd shows that the Fe-hydride is part of a H-bonding network with the nearby bridging azadithiolate to facilitate fast proton exchange and catalytic turnover

Diameter Dependent Electron Transfer Kinetics in Semiconductor–Enzyme Complexes

Excited state electron transfer (ET) is a fundamental step for the catalytic conversion of solar energy into chemical energy. To understand the properties controlling ET between photoexcited nanoparticles and catalysts, the ET kinetics were measured for solution-phase complexes of CdTe quantum dots and Clostridium acetobutylicum [FeFe]-hydrogenase I (CaI) using time-resolved photoluminescence spectroscopy. Over a 2.0–3.5 nm diameter range of CdTe nanoparticles, the observed ET rate (<i>k</i>_ET) was sensitive to CaI concentration. To account for diameter effects on CaI binding, a Langmuir isotherm and two geometric binding models were created to estimate maximal CaI affinities and coverages at saturating concentrations. Normalizing the ET kinetics to CaI surface coverage for each CdTe diameter led to <i>k</i>_ET values that were insensitive to diameter, despite a decrease in the free energy for photoexcited ET (Δ<i>G</i>_ET) with increasing diameter. The turnover frequency (TOF) of CaI in CdTe–CaI complexes was measured at several molar ratios. Normalization for diameter-dependent changes in CaI coverage showed an increase in TOF with diameter. These results suggest that <i>k</i>_ET and H₂ production for CdTe–CaI complexes are not strictly controlled by Δ<i>G</i>_ET and that other factors must be considered

Influence of growth factors on the <i>in vitro</i> hydrogen evolution activities in cells harboring pHydEFd-HydA1 and pHydFHydG.

<p>Hydrogenase activities (reported in units of nmol hydrogen evolved, ml⁻¹ of culture, min⁻¹) in solubilized whole-cells were measured as described in materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035886#s4" target="_blank">methods</a>. (A). The effect of the shaker rotation speed (RPM) during the aerobic growth phase was tested for 100 ml or 1 L culture volumes grown in. 250 ml or 2 L baffled flasks, respectively. The optimal rate was for both culture volumes was ∼300–350 RPM. (B). The effect of ferric ammonium citrate (FEC) concentration. Optimal levels were 2–2.5 mM. (C). Effect of IPTG concentration. Optimal levels of IPTG were 0.4 mM, lower than the 1.5 mM used previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035886#pone.0035886-Evans1" target="_blank">[13]</a>. (D). Effect of substituting Ampicillin with Carbenicillin during all stages of growth. Addition of Carbenicillin at 200 µg ml⁻¹ was required for peak hydrogenase activity levels.</p

SDS-PAGE analysis of Fd-HydA1 expression, TEV digestion and HydA1 purification.

<p>(A). A comparison of the expression levels of N-terminal and C-terminal Fd-HydA1 fusions by separation on SDS-PAGE. Lanes show (left to right) protein size marker, SM, with sizes in kDa, and increasing amounts (0.5-to-4 µl) of total protein from cells expressing either the C-terminal (Fd C) or N-terminal (Fd N) Fd-HydA1 fusions. The location of ∼60 kDa Fd-HydA1 fusion, and 53 kDa HydG as an internal loading control are marked. The difference of Fd position on Fd-HydA1 expression levels was evident as a more intense brown color of the cell-lysates (inset image, top) harboring the N-terminal (right) versus C-terminal (left) Fd fusion. A Western Blot performed with antibodies to the StrepII-tag is shown (inset image, bottom) where the N-terminal fusion (right) exhibits a more intense band than the C-terminal fusion (left). (B). Analysis of TEV digestions of the N-terminal Fd-HydA1 fusion (30 µg) mixed with 1, 2, 4, or 8 µg of TEV (lanes 1–4, respectively). Lanes 2 and 3 show a partial digestion. The locations of Fd-HydA1 (60 kDa), HydA1 (50 kDa) and Fd (10 kDa) are indicated on the right. The optimal amount of TEV for complete digestion was 4 µg (shown in lane 3). Protein size-marker, SM, with sizes in kDa. (C). TEV digestions of DEAE pooled fractions containing Fd-HydA1 and separated on SDS-PAGE. The same w/w ratio of TEV to Fd-HydA1 was used as for purified Fd-HydA1 (4 µg TEV with 30 µg of DEAE pool). Lane 1, complete TEV digest; Lane 2, partial TEV digest; Lane 3, no TEV. Bands corresponding to Fd-HydA1 (60 kDa), HydA1 (50 kDa) and Fd (10 kDa) are identified on the right. HydG is identified as a loading control. (D). Analysis of HydA1 purity by SDS-PAGE. Protein size marker, SM, with sizes in kDa. Lanes 1–4 contain 1.4, 4, 6.8, and 12.3 µg, respectively, of purified HydA1 at >99% purity.</p

HydA1 purification yields and specific activities from various expression hosts.

a<p>HydA1 specific activities for hydrogen evolution by MV assay; 1 U = µmol hydrogen min⁻¹ mg⁻¹.</p>b<p>HydA1 mg L⁻¹ of cell culture.</p>c<p>Iron content as mol iron (mol HydA1)⁻¹; NR, not reported.</p

Buffers used for protein purification.

<p>Buffers used for protein purification.</p

A) pH activity profile of CaHydA WT (full line) and C298D (dashed line).

<p>Hydrogen evolution activity was assayed in the pH range 5.0–9.0 using appropriate buffering agents at 37°C, with 10 mM reduced methyl viologen as artificial electron donor. Relative activity was calculated as the ratio with the maximum activity. <b>B) Dixon-Webb Plot for Log </b><b><i>k</i></b><b>_cat</b><b><i>vs</i></b><b> pH activity profile of CaHydA WT (full line) and C298D (dashed line).</b> The datapoints at higher pH (8.5–9.0) were not fitted to the Dixon-Webb Model since effects due to low proton concentration compared to the enzyme concentration (nM) are expected to occur in this region.</p

Characterization of Photochemical Processes for H₂ Production by CdS Nanorod–[FeFe] Hydrogenase Complexes

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 H₂ 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 H₂. Self-assembly of CdS with CaI was driven by electrostatics, demonstrated as the inhibition of ferredoxin-mediated H₂ evolution by CaI. Production of H₂ 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 H₂ 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, H₂ production was limited by the rate of photon absorption (∼1 ms^–1) and not by the turnover of CaI. Complexes were capable of H₂ production for up to 4 h with a total turnover number of 10⁶ before photocatalytic activity was lost. This loss correlated with inactivation of CaI, resulting from the photo-oxidation of the CdS capping ligand MPA