Developing high-affinity, oxygen-insensitive [NiFe]-hydrogenases as biocatalysts for energy conversion

Abstract

The splitting of hydrogen (H2) is an energy-yielding process, which is important for both biological systems and as a means of providing green energy. In biology, this reaction is mediated by enzymes called hydrogenases, which utilise complex nickel and iron cofactors to split H2 and transfer the resulting electrons to an electron-acceptor. These [NiFe]-hydrogenases have received considerable attention as catalysts in fuel cells, which utilise H2 to produce electrical current. [NiFe]-hydrogenases are a promising alternative to the platinum-based catalysts that currently predominate in fuel cells due to the abundance of nickel and iron, and the resistance of some family members to inhibition by gases, including carbon monoxide, which rapidly poison platinum-based catalysts. However, the majority of characterised [NiFe]-hydrogenases are inhibited by oxygen (O2), limiting their activity and stability. We recently reported the isolation and characterisation of the [NiFe]-hydrogenase Huc from Mycobacterium smegmatis, which is insensitive to inhibition by O2 and has an extremely high affinity, making it capable of oxidising H2 in air to below atmospheric concentrations. These properties make Huc a promising candidate for the development of enzyme-based fuel cells (EBFCs), which utilise H2 at low concentrations and in impure gas mixtures. In this review, we aim to provide context for the use of Huc for this purpose by discussing the advantages of [NiFe]-hydrogenases as catalysts and their deployment in fuel cells. We also address the challenges associated with using [NiFe]-hydrogenases for this purpose, and how these might be overcome to develop EBFCs that can be deployed at scale.

Keywords: [NiFe]-hydrogenase; electrocatalysis; enzyme-based fuel cells; hydrogen.

Figure 1.. The catalytic structure of [FeFe] and [NiFe]-hydrogenases.

(a) The architecture of the catalytic clusters of [NiFe] and [FeFe]-hydrogenases. Lig = H₂ or OH depending of the state of the catalytic cluster. (b) The CryoEM structure of the complex [NiFe]-hydrogenase Huc from M. smegmatis (PDB ID = 7UUS). A single catalytic unit consisting of HucS and HucL subunits (HucSL) is indicated, as are the [NiFe]-catalytic cluster, electron transferring [3Fe-4S] clusters, and electron accepting menaquinone.

Figure 2.. The application of [NiFe]-hydrogenases in fuel cell development.

(a) A simplified schematic of the general design for a PEMFC. (b) A simplified schematic for the general design of a membrane-less [NiFe]-hydrogenase EBFC, similar to those described by Xu and Armstrong [14]. (c) Cyclic voltammograms showing the current produced by Huc in the presence of different concentrations of H₂, adapted from Grinter and Kropp et al. [40] (d) Cyclic voltammograms showing the performance of Hyd1 from E. coli (EcHyd1) and Bilirubin oxidase from Myrothecium verrucaria (MvBO) in different H₂ gas mixtures. Oxidative inactivation of EcHyd1 is observed at potentials of greater than ∼0.0 V vs SHE, adapted with permission from Wait et al. [56].

Figure 3.. The structure of the [NiFe]-hydrogenase core catalytic complex.

The structure of core large and small catalytic subunits of [NiFe]-hydrogenases (left), and the arrangement of catalytic [NiFe] and electron transferring [FeS] cofactors present in the [NiFe]-hydrogenases large and small subunits (right). In this figure, the large and small subunits of Huc are shown for illustrative purposes (PDB ID = 7UUR).

Figure 4.. The complex structural of native [NiFe]-hydrogenases limits electron transfer to electrodes.

(a) Structures of examples of [NiFe]-hydrogenases that form parts of larger multisubunit complexes. (b) A schematic showing the optimal orientation of a [NiFe]-hydrogenase, for electron transfer, when associated with an electrode. (c) A cutaway surface view of the Huc complex structure shows that the electron-acceptor sites, with menaquinone bound, are located in an internal chamber of the complex, which shields them from the bulk solvent.