Structural Insight into the Complex of Ferredoxin and [FeFe] Hydrogenase from Chlamydomonas reinhardtii

The transfer of photosynthetic electrons by the ferredoxin PetF to the [FeFe] hydrogenase HydA1 in the microalga Chlamydomonas reinhardtii is a key step in hydrogen production. Electron delivery requires a specific interaction between PetF and HydA1. However, because of the transient nature of the electron‐transfer complex, a crystal structure remains elusive. Therefore, we performed protein–protein docking based on new experimental data from a solution NMR spectroscopy investigation of native and gallium‐substituted PetF. This provides valuable information about residues crucial for complex formation and electron transfer. The derived complex model might help to pinpoint residue substitution targets for improved hydrogen production.     

Electrocatalytic investigations of a tri-iron cluster towards hydrogen evolution and relevance to [FeFe]-hydrogenase

Electrochemical investigations of a tri-iron cluster, [Fe₃(μ₃-S)₂(CO)₉] (designated as Fe₃CO thereafter), which possesses a core of [Fe^IIFe^IFe^I], is described. In 0.5 M [NBu₄]BF₄/dichloromethane, the cluster exhibits a fairly reversible redox process at −1.03 V and irreversible reduction wave at ca. −1.75 V. The latter is attributed to the reductions of both the decomposed product of the monoanion, [Fe₃CO]⁻, and the isomer of this anion. This cluster catalyses proton reduction with the presence of acid HBF₄·Et₂O in dichloromethane. Possible mechanisms were proposed to elucidate its electrochemistry and electrocatalytic behaviours on proton reduction. Digital simulations were performed to verify the proposed mechanisms and kinetic parameters were generated at our best estimation in the simulations. The simulated cyclic voltammograms fit well with the experimentally observed ones, which largely supports the proposed mechanisms.     

Application of a Synthetic Ferredoxin-Inspired [4Fe4S]-Peptide Maquette as the Redox Partner for an [FeFe]-Hydrogenase

‘Bacterial-type’ ferredoxins host a cubane [4Fe4S]^2+/+ cluster that enables these proteins to mediate electron transfer and facilitate a broad range of biological processes. Peptide maquettes based on the conserved cluster-forming motif have previously been reported and used to model the ferredoxins. Herein we explore the integration of a [4Fe4S]-peptide maquette into a H₂-powered electron transport chain. While routinely formed under anaerobic conditions, we illustrate by electron paramagnetic resonance (EPR) analysis that these maquettes can be reconstituted under aerobic conditions by using photoactivated NADH to reduce the cluster at 240 K. Attempts to tune the redox properties of the iron-sulfur cluster by introducing an Fe-coordinating selenocysteine residue were also explored. To demonstrate the integration of these artificial metalloproteins into a semi-synthetic electron transport chain, we utilize a ferredoxin-inspired [4Fe4S]-peptide maquette as the redox partner in the hydrogenase-mediated oxidation of H₂.     

Mechanism and Application of the Catalytic Reaction of [NiFe] Hydrogenase: Recent Developments

Hydrogenases (H₂ase) catalyze the oxidation of dihydrogen and the reduction of protons with remarkable efficiency, thereby attracting considerable attention in the energy field due to their biotechnological potential. For this simple reaction, [NiFe] H₂ase has developed a sophisticated but intricate mechanism with the heterolytic cleavage of dihydrogen, where its Ni−Fe active site exhibits various redox states. Recently, new spectroscopic and crystal structure studies of [NiFe] H₂ases have been reported, providing significant insights into the catalytic reaction mechanism, hydrophobic gas-access tunnel, proton-transfer pathway, and electron-transfer pathway of [NiFe] H₂ases. In addition, [NiFe] H₂ases have been shown to play an important role in biofuel cell and solar dihydrogen production. This concept provides an overview of the biocatalytic reaction mechanism and biochemical application of [NiFe] H₂ases based on the new findings.     

Artificial Maturation of the Highly Active Heterodimeric [FeFe] Hydrogenase from Desulfovibrio desulfuricans ATCC 7757

Hydrogenases catalyze the reduction of protons and oxidation of molecular hydrogen with high turnover frequencies and low overpotentials under ambient conditions. The heterodimeric [FeFe] hydrogenase from Desulfovibrio desulfuricans has an exceptionally high activity, and can be purified aerobically in an oxygen-stable inactive state. Recently, it was demonstrated that monomeric [FeFe] hydrogenases produced recombinantly in Escherichia coli can be artificially maturated by simply incubating the inactive “apo” enzymes with the synthetic [2Fe] cofactor mimic [Fe₂(adt)(CO)₄(CN)₂]²⁻. Here, we use the same technique to produce the heterodimeric “apo” hydrogenase from D. desulfuricans in E. coli with a high yield and purity, and maturate the “apo” enzyme with [Fe₂(adt)(CO)₄(CN)₂]²⁻ to generate fully active “holo” enzyme. Interestingly, the rate of the artificial maturation process with D. desulfuricans is significantly slower than that for all other hydrogenases tested so far. The artificially maturated enzyme is spectroscopically and electrochemically identical to the native enzyme and shows high rates of hydrogen production (3700 s⁻¹) and hydrogen oxidation (63,000 s⁻¹). We expect that our highly efficient production method will facilitate future studies of this enzyme and other related [FeFe] hydrogenases from Desulfovibrio species.     

The [4Fe-4S]-Cluster of HydF is not Required for the Binding and Transfer of the Diiron Site of [FeFe]-Hydrogenases

The active site of [FeFe]-hydrogenases contains a cubane [4Fe-4S]-cluster and a unique diiron cluster with biologically unusual CO and CN⁻ ligands. The biogenesis of this diiron site, termed [2Fe_H], requires the maturation proteins HydE, HydF and HydG. During the maturation process HydF serves as a scaffold protein for the final assembly steps and the subsequent transfer of the [2Fe_H] precursor, termed [2Fe_P], to the [FeFe]-hydrogenase. The binding site of [2Fe_P] in HydF has not been elucidated, however, the [4Fe-4S]-cluster of HydF was considered as a possible binding partner of [2Fe_P]. By targeting individual amino acids in HydF from Thermosipho melanesiensis using site directed mutagenesis, we examined the postulated binding mechanism as well as the importance and putative involvement of the [4Fe-4S]-cluster for binding and transferring [2Fe_P]. Surprisingly, our results suggest that binding or transfer of [2Fe_P] does not involve the proposed binding mechanism or the presence of a [4Fe-4S]-cluster at all.     

Designed Surface Residue Substitutions in [NiFe] Hydrogenase that Improve Electron Transfer Characteristics

Photobiological hydrogen production is an attractive, carbon-neutral means to convert solar energy to hydrogen. We build on previous research improving the Alteromonas macleodii “Deep Ecotype” [NiFe] hydrogenase, and report progress towards creating an artificial electron transfer pathway to supply the hydrogenase with electrons necessary for hydrogen production. Ferredoxin is the first soluble electron transfer mediator to receive high-energy electrons from photosystem I, and bears an electron with sufficient potential to efficiently reduce protons. Thus, we engineered a hydrogenase-ferredoxin fusion that also contained several other modifications. In addition to the C-terminal ferredoxin fusion, we truncated the C-terminus of the hydrogenase small subunit, identified as the available terminus closer to the electron transfer region. We also neutralized an anionic patch surrounding the interface Fe-S cluster to improve transfer kinetics with the negatively charged ferredoxin. Initial screening showed the enzyme tolerated both truncation and charge neutralization on the small subunit ferredoxin-binding face. While the enzyme activity was relatively unchanged using the substrate methyl viologen, we observed a marked improvement from both the ferredoxin fusion and surface modification using only dithionite as an electron donor. Combining ferredoxin fusion and surface charge modification showed progressively improved activity in an in vitro assay with purified enzyme.     

[FeFe]-Hydrogenase Abundance and Diversity along a Vertical Redox Gradient in Great Salt Lake,USA

The use of [FeFe]-hydrogenase enzymes for the biotechnological production of H₂ or other reduced products has been limited by their sensitivity to oxygen (O₂). Here, we apply a PCR-directed approach to determine the distribution, abundance, and diversity of hydA gene fragments along co-varying salinity and O₂ gradients in a vertical water column of Great Salt Lake (GSL), UT. The distribution of hydA was constrained to water column transects that had high salt and relatively low O₂ concentrations. Recovered HydA deduced amino acid sequences were enriched in hydrophilic amino acids relative to HydA from less saline environments. In addition, they harbored interesting variations in the amino acid environment of the complex H-cluster metalloenzyme active site and putative gas transfer channels that may be important for both H₂ transfer and O₂ susceptibility. A phylogenetic framework was created to infer the accessory cluster composition and quaternary structure of recovered HydA protein sequences based on phylogenetic relationships and the gene contexts of known complete HydA sequences. Numerous recovered HydA are predicted to harbor multiple N- and C-terminal accessory iron-sulfur cluster binding domains and are likely to exist as multisubunit complexes. This study indicates an important role for [FeFe]-hydrogenases in the functioning of the GSL ecosystem and provides new target genes and variants for use in identifying O₂ tolerant enzymes for biotechnological applications.     

A CMP-based [FeFe]-hydrogenase dual-functional biomimetic system for photocatalytic hydrogen evolution coupled with degradation of tetracycline

A novel bio-inspired conjugated microporous polymer (CMP) had been designed, synthesized and characterized. The mimics of [FeFe]-hydrogenase active sites were covalently attached to the CMP skeleton, which facilitates charge transfer between the light-harvesting moiety and active sites, and exhibited high performance in visible-light photocatalytic hydrogen evolution (2120 μmol·h⁻¹·g⁻¹) in an aqueous solution. The flower-like morphology, covalently linked framework and the “single active-site” effect derived from the porous skeleton were deemed to go a long way toward boosting the properties above. Interestingly, when the electron sacrifice agent (triethanolamine) was replaced by tetracycline, the CMP-based photocatalyst maintained the capability of photocatalytic hydrogen production (370 μmol·h⁻¹·g⁻¹) and realized efficient photodegradation of tetracycline simultaneously. This work provides a heuristic green dual-function strategy for constructing a sustainable and efficient photocatalytic system for hydrogen evolution with concurrent antibiotic residue degradation.     

Crystal Structure of HydG from Carboxydothermus hydrogenoformans: A Trifunctional [FeFe]‐Hydrogenase Maturase

The structure of the radical S‐adenosyl‐L ‐methionine (SAM) [FeFe]‐hydrogenase maturase HydG involved in CN^?/CO synthesis is characterized by two internal tunnels connecting its tyrosine‐binding pocket with the external medium and the C‐terminal Fe₄S₄ cluster‐containing region. A comparison with a tryptophan‐bound NosL structure suggests that substrate binding causes the closing of the first tunnel and, along with mutagenesis studies, that tyrosine binds to HydG with its amino group well positioned for H‐abstraction by SAM. In this orientation the dehydroglycine (DHG) fragment caused by tyrosine Cα?Cβ bond scission can readily migrate through the second tunnel towards the C‐terminal domain where both CN^? and CO are synthesized. Our HydG structure appears to be in a relaxed state with its C‐terminal cluster CysX₂CysX₂₂Cys motif exposed to solvent. A rotation of this domain coupled to Fe₄S₄ cluster assembly would bury its putatively reactive unique Fe ion thereby allowing it to interact with DHG.     

Preparation of Novel Pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine Sulfonamides and Their Experimental and Computational Biological Studies

Pyrazolo[4,3-e]tetrazolo[1,5-b][1,2,4]triazine sulfonamides constitute a novel class of heterocyclic compounds with broad biological activity, including anticancer properties. Investigated in this study, MM-compounds (MM134, MM136, MM137, and MM139) exhibited cytotoxic and proapoptotic activity against cancer cell lines (BxPC-3, PC-3, and HCT-116) in nanomolar concentrations without causing cytotoxicity in normal cells (L929 and WI38). In silico predictions indicate that tested compounds exhibit favorable pharmacokinetic profiles and may exert anticancer activity through the inhibition of BTK kinase, the AKT-mTOR pathway and PD1-PD-L1 interaction. Our findings point out that these sulfonamide derivatives may constitute a source of new anticancer drugs after optimization.     

Theoretical Spectroscopy of the NiII Intermediate States in the Catalytic Cycle and the Activation of [NiFe] Hydrogenases

[NiFe] hydrogenases catalyze the reversible oxidation of dihydrogen. The corresponding catalytic cycle involves a formidable number of redox states of the Ni‐Fe active site; these can be distinguished experimentally by the IR stretching frequencies of their CN and CO ligands coordinated to iron. These spectroscopic fingerprints serve as sensitive probes for the intrinsic electronic structure of the metal core and, indirectly, for the structural composition of the active site. In this study, density functional theory (DFT) was used to calculate vibrational frequencies, by focusing on the EPR‐silent intermediate states that contain divalent metal centers. By using the well‐characterized Ni‐C and Ni‐B states as references, we identified candidates for the Ni‐SI_r, Ni‐SI_a, and Ni‐R states by matching the predicted relative frequency shifts with experimental results. The Ni‐SI_r and Ni‐SI_a states feature a water molecule loosely bound to nickel and a formally vacant bridge. Both states are connected to each other through protonation equilibria; that is, in the Ni‐SI_a state one of the terminal thiolates is protonated, whereas in Ni‐SI_r this thiolate is unprotonated. For the reduced Ni‐R state two feasible models emerged: in one, H₂ coordinates side‐on to nickel, and the second features a hydride bridge and a protonated thiolate. The Ni‐SU state remains elusive as no unequivocal correspondence between the experimental data and calculated frequencies of the models was found, thus indicating that a larger structural rearrangement might occur upon reduction from Ni‐A to Ni‐SU and that the bridging ligand might dissociate.     

Computational studies of the gramicidin channel

Ion channels are highly specific membrane-spanning protein structures which serve to facilitate the passage of selected ions across the lipid barrier. In the past decade, molecular dynamics simulations based on atomic models and realistic microscopic interactions with explicit solvent and membrane lipids have been used to gain insight into the function of these complex systems. These calculations have considerably expanded our view of ion permeation at the microscopic level. This Account will mainly focus on computational studies of the gramicidin A channel, one of the simplest and best characterized molecular pore.     

X-ray and molecular modelling studies of 4-[N-alkylamino]azobenzene dyes

4-[N,N-Bisalkyl]amino-2′-chloro-4′-nitroazobenzenes were recrystallized from acetone in either triclinic or monoclinic cells with the space group P-1 or P2₁/c. The asymmetric unit cell of dyes having at least one N-cyanoethyl group contained two molecules that were symmetrically unequivalent. The aromatic rings in the azobenzene skeleton were essentially planar with respect to the plane of the azo group, although the C6–C1–N1–N2 torsion angle was 13.2° when the N,N-dicyanoethyl group was employed. X-ray studies were used as a basis for assessing the utility of nonlocal DFT calculations in predicting the equilibrium molecular geometry and solvatochromic properties of the compounds using MM3/ZINDO-S and the COSMO Solvation Model. Although B3LYP and PBE energy functionals were comparable in predicting bond lengths, PBE was slightly better than B3LYP in predicting torsion angles. Furthermore, the dipolarity/polarizability index (π¹) was the preferred solvent parameter for predicting the effects of solvents on λ_max.