From protein engineering to artificial enzymes - biological and biomimetic approaches towards sustainable hydrogen production

Abstract

Hydrogen gas is used extensively in industry today and is often put forward as a suitable energy carrier due its high energy density. Currently, the main source of molecular hydrogen is fossil fuels via steam reforming. Consequently, novel production methods are required to improve the sustainability of hydrogen gas for industrial processes, as well as paving the way for its implementation as a future solar fuel. Nature has already developed an elaborate hydrogen economy, where the production and consumption of hydrogen gas is catalysed by hydrogenase enzymes. In this review we summarize efforts on engineering and optimizing these enzymes for biological hydrogen gas production, with an emphasis on their inorganic cofactors. Moreover, we will describe how our understanding of these enzymes has been applied for the preparation of bio-inspired/-mimetic systems for efficient and sustainable hydrogen production.

Fig. 1. [NiFe], [FeFe] and [Fe] hydrogenases. (a) The “apo” form of Chlamydomonas reinhardtii HydA1 ([FeFe] hydrogenase; PDB: ; 3LX4); (b) Clostridium pasteurianum CpI ([FeFe hydrogenase]; PDB: ; 4XDC); (c) Allochromatium vinosum [NiFe] hydrogenase (PDB: ; 3MYR); (d) Methanocaldococcus jannaschii [Fe] hydrogenase (PDB: ; 3DAG). Protein backbones are shown as solid ribbons and coloured by chain, and metal cofactors are shown as space-filling models and coloured by element (structures drawn using BIOVIA Discovery Studio Visualizer). A schematic representation of the active site of the different subclasses is shown below the respective enzyme structure. Heteroatom colour coding: S: yellow; Fe: indigo; Ni: dark blue; O: red; N: blue; P: orange.

Fig. 2. Skeletal representation of the catalytic cycle of the [NiFe] hydrogenase including the Ni_L-state.

Fig. 3. Skeletal representation of the catalytic cycle of the [FeFe] hydrogenase, adapted from Sommer et al.

Fig. 4. Schematic representation of the catalytic cycle of the [Fe] hydrogenase.

Fig. 5. Strategies to control catalytic bias and improve tolerance to O₂ in hydrogenases. Protein engineering allows tuning proton and electron delivery and controlling the access of O₂ to the active site.

Fig. 6. Engineering of the uptake hydrogenase HupSL in Nostoc punctiforme for hydrogen production. Left: N. punctiforme expressing C12P HupS (C12PNp strain) produces H₂ in addition to the background production from nitrogenase activity (ΔHupNp strain). Right: the C12P mutation induces a [4Fe–4S] → [3Fe–4S] cluster conversion in the proximal position in HupS, facilitating reverse electron flow towards the active site. The figure was adapted from Raleiras et al. with permission from the Royal Society of Chemistry.

Fig. 7. The coordination sphere of the [NiFe] site in the D. fructosovorans hydrogenase (PDB: ; 1YRQ). Through site-directed mutagenesis, valine-74 has been shown to have a role in the gas accessibility to the active site, possibly through a gating action. Blue ribbon: large subunit; green ribbon: small subunit; heteroatom colour coding as in Fig. 1.

Fig. 8. Structural comparison between proximal [FeS] clusters in O₂-tolerant and O₂-sensitive [NiFe] hydrogenases. Left: the proximal [4Fe–3S] cluster in the R. eutropha MBH (PDB: ; 3RGW), involved in the direct detoxification of O₂. Right: the proximal [4Fe–4S] cluster in the D. fructosovorans hydrogenase (PDB: ; 1YRQ). Cysteine residues directly coordinating the clusters are shown. Heteroatom colour coding as in Fig. 1.

Fig. 9. Proton and electron transfer chains in the C. pasteurianum [FeFe] hydrogenase (PDB: ; 3C8Y). Highlighted residues have been identified as part of a hydrogen bond network transporting protons between the protein surface and the active site (H-cluster). Heteroatom colour coding as in Fig. 1.

Fig. 10. Schematic representation of the insertion of a metallic cofactor in a scaffold highlighting the potential contributions of the outer coordination sphere.

Fig. 11. (a) Structure of the active site of myoglobin (PDB ID: 1YOI) showing relevant histidine residues and structure of Co-protoporphyrin IX (CoP, 1). (b) Cobalt catalysts (cobaloxime derivatives) inserted into a myoglobin matrix. (c) Scheme of the Ru–Fd–Co, 4 biohybrid, based on the structure of ferredoxin (PDB ID: ; 1A70) with a potential pathway for electron transfer from the PS to the catalyst. (d) Preparation of the artificial hydrogenase containing a {μ-(SCH₂)₂CHCOR}Fe₂(CO)₆ (7) moiety within a Q96C apo-nitrobindin (apoNB) (PDB ID for NB: ; 2A13).

Fig. 12. Schematic representation of the (a) cytochrome c-556 fragment containing a [(μ-S₂)Fe₂(CO)₆] moiety and a ruthenium-based photosensitizing unit, (b) helical nonadecapeptide containing a [(μ-S(CH₂)₃S)Fe₂(CO)₆] motif, and (c) peptide containing an unnatural amino acid linked to a [(μ-pdt)Fe₂(CO)₆] (10) complex and (d) formation of a heterobimetallic cluster in a heptapeptide construct.

Fig. 13. Schematic representation of mononuclear nickel complexes bearing functionalized PR2NR′2 ligands.

Fig. 14. Schematic representation of CoGly–Gly–His (CoATCUN, 20) and CoMP11-Ac, 21, and the structure of cytochrome c-552 (PDB ID: ; 1YNR) (Ht c-552, 22 also showing the native Met-ligand).

Fig. 15. Schematic representation of an artificial system for photocatalytic H₂ production, including [(μ-pdt)Fe₂(CO)₅(CNR)] isocyanide complexes. 23, linked to a water soluble PAA polymer and MPA-CdSe QDs.

Fig. 16. Schematic representation of a diiron mimic encapsulated in a cyclodextrin unit, and the structure of the α (n = 0), β (n = 1), and γ (n = 2) cyclodextrin units.

Fig. 17. Schematic representation of artificial maturation of the maturase HydF and the hydrogenase enzyme, HydA.

Fig. 18. FTIR spectra with normalized intensities of selected forms of semi-synthetic HydA, loaded with synthetic co-factors 30–32 (30–32-HydA1), compared to a form of HydA containing the native co-factor (CrHydA1). The figure was adapted from Berggren et al., Nature, 2013.

Fig. 19. Overview of synthetic complexes screened for insertion into [FeFe] hydrogenases.

Fig. 20. Comparison of 30-CpI (carbon atoms in marine) superposed on stick models of 31-CpI (magenta), 32-CpI (green) and 38-CpI (yellow). Dashed lines indicate potential proton transfer pathways or potential interactions of the [2Fe] subsite with the protein. Amino acid numbering as in the structure of native CpI. Adapted from Esselborn et al. with permission from the Royal Society of Chemistry.

Fig. 21. FTIR spectra with normalized intensities of selected forms of semi-synthetic HydF, loaded with synthetic co-factors 30–32 (30–32-HydF), compared to a form of HydF containing the native co-factor (CaHydF). The figure was adapted from Berggren et al., Nature, 2013.

Fig. 22. Complexes 46 and 47 are inserted into the apo-[Fe] hydrogenase following the removal of two monodentate ligands (CO and I^–). Substituents on the pyridine ring lacking in 46 and 47 as compared to the native FeGP cofactor indicated in red.