Retuning Nature's Potential by Active Site Engineering and Electrochemical Control

In this project, the pivotal enzyme in the Electrochemical Leaf (e-Leaf) (a new concept/platform in which tandem catalysis by multi-enzyme cascades is electrochemically driven and controlled), will be engineered to change its natural redox properties with an overall aim of elucidating how biology tunes the reduction potential of enzyme cofactors. Unravelling nature's strategies that enable redox enzymes to carry out reactions that would otherwise be unfeasible for the cofactor alone (i.e. not bound to the enzyme), will ultimately be exploited to engineer bespoke enzymes with altered reduction potentials for new biocatalytic systems.

Ferredoxin NADP+ reductase (FNR) is a key enzyme in photosynthesis that connects the light reactions to the Calvin-Benson enzyme cascade by mobilising light-energised electrons as hydride on NADPH (biology's mobile electron carrier molecule). In the e-Leaf, aspects of photosynthesis are paralleled: sunlight is replaced by electricity, which provides the energised electrons, and the Calvin-Benson enzyme cascade can be replaced with any biosynthetic enzyme cascade of choice.

Central to the concept is FNR, immobilised in a porous metal oxide electrode where, under potential control, electrons can be directly supplied to and from its active site flavin cofactor, enabling it to catalyse the interconversion of NADP+/NADPH.

Preliminary results show that a single amino acid change at FNR's active site leads to a shift in the reduction potential of the flavin towards a more oxidative voltage. FNR will be engineered to probe the mechanism by which this tuning occurs with guidance from computational modelling to predict mutations important to the mechanism. In addition, native (FAD) will be replaced by different flavin analogues, both in the WT enzyme and in previously engineered variants. Reduction potentials and catalytic activity will be monitored and investigated electrochemically.

This project will lead to fundamental understanding and will ultimately expand the scope of the e-Leaf technology because the engineered FNR variants will be used to investigate and drive biosynthetic cascades. Knowledge gained will be applied to other enzyme systems, for example changing a unidirectional enzyme into a bidirectional one by tuning its potential to be very close to that of its overall reaction so that only a small change either side of this redox couple, will result in the flow of catalytic current.

Moreover, learning how nature achieves this level of fine control over reduction potentials may impact the design of molecular complexes and will be exploited to engineer enzymes with altered reduction potentials for new bio-electrochemical systems.

This project falls within several of the UKRI's research priorities including, Understanding the Rules of Life, Transformative Technologies, Engineering, Bioscience for renewable resources and Clean Growth, Manufacturing the Future (Building a Green Future), Chemical Biology and Biological chemistry, Electrochemical Sciences, and Catalysis.