From Protein Film Electrochemistry to Nanoconfined Enzyme Cascades and the Electrochemical Leaf

Abstract

Protein film electrochemistry (PFE) has given unrivalled insight into the properties of redox proteins and many electron-transferring enzymes, allowing investigations of otherwise ill-defined or intractable topics such as unstable Fe–S centers and the catalytic bias of enzymes. Many enzymes have been established to be reversible electrocatalysts when attached to an electrode, and further investigations have revealed how unusual dependences of catalytic rates on electrode potential have stark similarities with electronics. A special case, the reversible electrochemistry of a photosynthetic enzyme, ferredoxin-NADP+ reductase (FNR), loaded at very high concentrations in the 3D nanopores of a conducting metal oxide layer, is leading to a new technology that brings PFE to myriad enzymes of other classes, the activities of which become controlled by the primary electron exchange. This extension is possible because FNR-based recycling of NADP(H) can be coupled to a dehydrogenase, and thence to other enzymes linked in tandem by the tight channelling of cofactors and intermediates within the nanopores of the material. The earlier interpretations of catalytic wave-shapes and various analogies with electronics are thus extended to initiate a field perhaps aptly named “cascade-tronics”, in which the flow of reactions along an enzyme cascade is monitored and controlled through an electrochemical analyzer. Unlike in photosynthesis where FNR transduces electron transfer and hydride transfer through the unidirectional recycling of NADPH, the “electrochemical leaf” (e-Leaf) can be used to drive reactions in both oxidizing and reducing directions. The e-Leaf offers a natural way to study how enzymes are affected by nanoconfinement and crowding, mimicking the physical conditions under which enzyme cascades operate in living cells. The reactions of the trapped enzymes, often at very high local concentration, are thus studied electrochemically, exploiting the potential domain to control rates and direction and the current–rate analogy to derive kinetic data. Localized NADP(H) recycling is very efficient, resulting in very high cofactor turnover numbers and new opportunities for controlling and exploiting biocatalysis.

Design principles for a nanoconfined enzyme cascade electrode via reaction–diffusion modelling

“…This current enhancement is observable even down to [NADP + ] of a few μM, which is consistent with experimental results that achieved a significant coupled current in the presence of a downstream enzyme. 1,2 At higher [NADP + ], the current reaches a transport-limited plateau (of E2 substrate from the bulk to the electrode outer surface), and at the highest [NADP + ] values (∼10 mM, i.e. when the bulk [NADP + ] is on the same order as the E2 substrate concentration) this plateau is exceeded due to the addition of the current from reduction of bulk [NADP + ].…”

“…Inspired by biological cascades, the 'electrochemical leaf' is a recently developed platform technology: an electrondriven, nanoconfined enzymatic cascade, immobilized within a porous indium tin oxide (ITO) electrode. [1][2][3][4] The key enzyme is ferredoxin-NADP + reductase (FNR), a component of the photosynthesis cascade. When FNR is directly in contact with a suitable electrode surface, it is electroactive, able to quasi-reversibly catalyse the 2e À interconversion of NADP + /NADPH, a recyclable redox cofactor.…”

Orientation-Controllable Enzyme Cascade on Electrode for Bioelectrocatalytic Chain Reaction

“…Importantly, we suggested how to strategically utilize this molecular binder to concurrently control bienzymatic chain reaction and DET process at the enzyme-electrode interface. We believe that the concept of integrating SBP-fusion technology and enzyme cascades may provide new opportunities for rational biosynthetic design of enzyme cascade-based bioelectrocatalytic system that holds promise for applications in electrical power generation, biomedical sensors, cofactor regeneration, production of value-added biochemicals, artificial photosynthesis, etc. − …”

Comprehensive structural, infrared spectroscopic and kinetic investigations of the roles of the active-site arginine in bidirectional hydrogen activation by the [NiFe]-hydrogenase ‘Hyd-2’ from Escherichia coli

“…It follows therefore that a reversible catalyst is always bidirectional, but a bidirectional catalyst may not necessarily display reversibility. 23 Looking to the outer coordination shell for further insight, a number of catalyticallyimportant, non-coordinating amino acids have been identied in various [NiFe]-hydrogenases, and two of these appear crucial for activity. One such residue is a glutamate [24][25][26] (E28/E14 in Hyd-1/Hyd-2 respectively), the carboxylate of which lies close (<4 Å) to the Ni-coordinating terminal cysteine C576/C546; 25,27 the other is a strictly conserved 'pendant' arginine 27,28 (R509/R479) that is located in a canopy immediately above the catalytic metals.…”