Harnessing electron flow in cells
Electron transfer is used by mitochondria and chloroplasts and in single-celled organisms such as bacteria. Electrons are moved from a donor (loses electrons and is oxidised) to an acceptor (gains electrons and is reduced). These reduction-oxidation (redox) reactions are a vital component of the energy cycle. Recent advances in single-molecule recording techniques have been successfully applied to larger architectures such as DNA. The EU-funded project SINGLE-BIOET (Single-molecule junction capabilities to map the electron pathways in redox bio-molecular architectures) explored sequential electron transfer pathways within an individual biomolecular structure. Copper in the bacterial blue copper azurin metalloprotein undergoes oxidation-reduction associated with its electron transport chain. Researchers modified positions on the molecule with entities that enhance single-molecule binding using site-directed mutagenesis. They created single-molecule junctions at these positions on the outer shell of the protein to explore dominant parameters in the electron transfer chain. Investigators have established the experimental technique to create single-molecule junctions. The site-directed mutagenesis results have been tested by fluorescence and electrochemical means. Conductance was measured in mutant and wild-type azurin, providing insight into both the use of the experimental technique and electron transport. Differences in conductance between wild and mutant types demonstrated the feasibility of modulating charge transport in nanoscale molecular devices by simple point-site modification of the biomolecular backbones. A site-directed mutagenesis scheme resulted in the final nine selected residues of the outer azurin structure. All purified mutants have been characterised for their activity as well as structural folding. The comparison of the wild-type transport and the mutant variants provided information on the pathways through the complex metalloprotein structure and technical details on the single-protein bridge formation. The single-molecule conductance measurement on the modified proteins showed less dispersion in comparison to the wild type. This indicates the more restricted protein orientation thanks to the stronger thiol attachment to the two junction electrodes. The single-protein lifetime also increased in the mutant cases, as measured by a newly implemented blinking tool. Lastly, the electrochemical gating response of the single-mutant wires deviated completely from its wild-type counterpart, proving the feasibility of bioengineering charge transport in a nanoscale biomolecular wire. In conclusion, the characterisation of the charge transport mechanisms will have major impact on the emerging field of bioelectronics. It will facilitate integration of biological structures as components of optoelectronic devices.
Keywords
Electron transport, bioelectronics, redox, SINGLE-BIOET, azurin metalloprotein
