Multistep enzyme-catalyzed cascade reactions are highly efficient in nature due to the confinement and concentration of the enzymes within nanocompartments. In this way, rates are exceptionally high, ...and loss of intermediates minimised. Similarly, extended enzyme cascades trapped and crowded within the nanoconfined environment of a porous conducting metal oxide electrode material form the basis of a powerful way to study and exploit myriad complex biocatalytic reactions and pathways. One of the confined enzymes, ferredoxin-NADP
reductase, serves as a transducer, rapidly and reversibly recycling nicotinamide cofactors electrochemically for immediate delivery to the next enzyme along the chain, thereby making it possible to energize, control and observe extended cascade reactions driven in either direction depending on the electrode potential that is applied. Here we show as proof of concept the synthesis of aspartic acid from pyruvic acid or its reverse oxidative decarboxylation/deamination, involving five nanoconfined enzymes.
Living organisms are characterized by the ability to process energy (all release heat). Redox reactions play a central role in biology, from energy transduction (photosynthesis, respiratory chains) ...to highly selective catalyzed transformations of complex molecules. Distance and scale are important: electrons transfer on a 1 nm scale, hydrogen nuclei transfer between molecules on a 0.1 nm scale, and extended catalytic processes (cascades) operate most efficiently when the different enzymes are under nanoconfinement (10 nm-100 nm scale). Dynamic electrochemistry experiments (defined broadly within the term "protein film electrochemistry," PFE) reveal details that are usually hidden in conventional kinetic experiments. In PFE, the enzyme is attached to an electrode, often in an innovative way, and electron-transfer reactions, individual or within steady-state catalytic flow, can be analyzed in terms of precise potentials, proton coupling, cooperativity, driving-force dependence of rates, and reversibility (a mark of efficiency). The electrochemical experiments reveal subtle factors that would have played an essential role in molecular evolution. This article describes how PFE is used to visualize and analyze different aspects of biological redox chemistry, from long-range directional electron transfer to electron/hydride (NADPH) interconversion by a flavoenzyme and finally to NADPH recycling in a nanoconfined enzyme cascade.
In living cells, redox chains rely on nanoconfinement using tiny enclosures, such as the mitochondrial matrix or chloroplast stroma, to concentrate enzymes and limit distances that nicotinamide ...cofactors and other metabolites must diffuse. In a chemical analogue exploiting this principle, nicotinamide adenine dinucleotide phosphate (NADPH) and NADP+ are cycled rapidly between ferredoxin–NADP+ reductase and a second enzyme—the pairs being juxtaposed within the 5–100 nm scale pores of an indium tin oxide electrode. The resulting electrode material, denoted (FNR+E2)@ITO/support, can drive and exploit a potentially large number of enzyme‐catalysed reactions.
In close quarters: Nicotinamide adenine dinucleotide phosphate (NADPH) and NADP+ are cycled rapidly between ferredoxin–NADP+ reductase (FNR) and a dehydrogenase enzyme (E2) within the 5–100 nm pores of an indium tin oxide electrode. The electrode mimics the nanoconfinement strategies of natural systems, such as the chloroplast stroma or mitochondria in living cells.
The redox state of the plastoquinone (PQ) pool in sulfur-deprived, H-2-producing Chlamydomonas reinhardtii cells was studied using single flash-induced variable fluorescence decay kinetics. During ...H-2 production, the fluorescence decay kinetics exhibited an unusual post-illumination rise of variable fluorescence, giving a wave-like appearance. The wave showed the transient fluorescence minimum at 60 ms after the flash, followed by a rise, reaching the transient fluorescence maximum at 1 s after the flash, before decaying back to the initial fluorescence level. Similar wave-like fluorescence decay kinetics have been reported previously in anaerobically incubated cyanobacteria but not in green algae. From several different electron and proton transfer inhibitors used, polymyxin B, an inhibitor of type II NAD(P)H dehydrogenase (NDA2), had the effect of eliminating the fluorescence wave feature, indicating involvement of NDA2 in this phenomenon. This was further confirmed by the absence of the fluorescence wave in the Delta nda2 mutant lacking NDA2. Additionally, Delta nda2 mutants have also shown delayed and diminished H-2 production (only 23% if compared with the wild type). Our results show that the fluorescence wave phenomenon in C. reinhardtii is observed under highly reducing conditions and is induced by the NDA2-mediated electron flow from the reduced stromal components to the PQ pool. Therefore, the fluorescence wave phenomenon is a sensitive probe for the complex network of redox reactions at the PQ pool level in the thylakoid membrane. It could be used in further characterization and improvement of the electron transfer pathways leading to H-2 production in C. reinhardtii.
Electron transport, mediated by NDA2 in H2-producing C. reinhardtii cells, shifts redox equilibria between the plastoquinone pool and PSII, and is observed as a transient fluorescence wave after a ...single flash.
Abstract
The redox state of the plastoquinone (PQ) pool in sulfur-deprived, H2-producing Chlamydomonas reinhardtii cells was studied using single flash-induced variable fluorescence decay kinetics. During H2 production, the fluorescence decay kinetics exhibited an unusual post-illumination rise of variable fluorescence, giving a wave-like appearance. The wave showed the transient fluorescence minimum at ~60 ms after the flash, followed by a rise, reaching the transient fluorescence maximum at ~1 s after the flash, before decaying back to the initial fluorescence level. Similar wave-like fluorescence decay kinetics have been reported previously in anaerobically incubated cyanobacteria but not in green algae. From several different electron and proton transfer inhibitors used, polymyxin B, an inhibitor of type II NAD(P)H dehydrogenase (NDA2), had the effect of eliminating the fluorescence wave feature, indicating involvement of NDA2 in this phenomenon. This was further confirmed by the absence of the fluorescence wave in the Δnda2 mutant lacking NDA2. Additionally, Δnda2 mutants have also shown delayed and diminished H2 production (only 23% if compared with the wild type). Our results show that the fluorescence wave phenomenon in C. reinhardtii is observed under highly reducing conditions and is induced by the NDA2-mediated electron flow from the reduced stromal components to the PQ pool. Therefore, the fluorescence wave phenomenon is a sensitive probe for the complex network of redox reactions at the PQ pool level in the thylakoid membrane. It could be used in further characterization and improvement of the electron transfer pathways leading to H2 production in C. reinhardtii.
Hydrogenase-1 (Hyd-1) from E. coli poses a conundrum regarding the properties of electrocatalytic reversibility and associated bidirectionality now established for many redox enzymes. Its excellent ...H2-oxidizing activity begins only once a substantial overpotential is applied, and it cannot produce H2. A major reason for its unidirectional behavior is that the reduction potentials of its electron-relaying FeS clusters are too positive relative to the 2H+/H2 couple at neutral pH; consequently, electrons held within the enzyme lack the energy to drive H2 production. However, Hyd-1 is O2-tolerant and even functions in air. Changing a tyrosine (Y) or threonine (T), located on the protein surface within 10 Å of the distal 4Fe-4S and medial 3Fe-4S clusters, to cysteine (C), allows site-selective attachment of a silver nanocluster (AgNC), the reduced or photoexcited state of which is a powerful reductant. The AgNC provides a new additional redox site, capturing externally supplied electrons with sufficiently high energy to drive H2 production. Assemblies of Y’227C (or T’225C) with AgNCs/PMAA (PMAA = polymethyl acrylate templating several AgNC) are also electroactive for H2 production at a TiO2 electrode. A colloidal system for visible-light photo-H2 generation is made by building the hybrid enzyme into a heterostructure with TiO2 and graphitic carbon nitride (g-C3N4), the resulting scaffold promoting uptake of electrons excited at the AgNC. Each hydrogenase produces 40 molecules of H2 per second and sustains 20% activity in air.
Reduction of CO2 and its direct entry into organic chemistry is achieved efficiently and in a highly visible way using a metal oxide electrode in which two enzyme catalysts, one for electrochemically ...regenerating reduced nicotinamide adenine dinucleotide phosphate and the other for assimilating CO2 and converting pyruvate (C3) to malate (C4), are entrapped within its nanopores. The resulting reversible electrocatalysis is exploited to construct a solar CO2 reduction/water-splitting device producing O2 and C4 with high faradaic efficiency.
Hydrogenase-1 (Hyd-1) from
poses a conundrum regarding the properties of electrocatalytic reversibility and associated bidirectionality now established for many redox enzymes. Its excellent H
...-oxidizing activity begins only once a substantial overpotential is applied, and it cannot produce H
. A major reason for its unidirectional behavior is that the reduction potentials of its electron-relaying FeS clusters are too positive relative to the 2H
/H
couple at neutral pH; consequently, electrons held within the enzyme lack the energy to drive H
production. However, Hyd-1 is O
-tolerant and even functions in air. Changing a tyrosine (Y) or threonine (T), located on the protein surface within 10 Å of the distal 4Fe-4S and medial 3Fe-4S clusters, to cysteine (C), allows site-selective attachment of a silver nanocluster (AgNC), the reduced or photoexcited state of which is a powerful reductant. The AgNC provides a new additional redox site, capturing externally supplied electrons with sufficiently high energy to drive H
production. Assemblies of Y'227C (or T'225C) with AgNCs/PMAA (PMAA = polymethyl acrylate templating several AgNC) are also electroactive for H
production at a TiO
electrode. A colloidal system for visible-light photo-H
generation is made by building the hybrid enzyme into a heterostructure with TiO
and graphitic carbon nitride (g-C
N
), the resulting scaffold promoting uptake of electrons excited at the AgNC. Each hydrogenase produces 40 molecules of H
per second and sustains 20% activity in air.
The co-localisation of cooperating enzymes within nanozones is crucial in biological catalytic processes to ensure fast and efficient catalysis. The Electrochemical Leaf is a novel electrode which ...mimics that principle. Entrapping two cooperating enzymes, Ferredoxin NADP+ reductase (FNR) and a NADPH-dependent dehydrogenase, within the nanopores of an Indium-Tin-Oxide (ITO) electrode produces a massive catalytic enhancement. Upon application of a small potential FNR catalyses the interconversion of NADP+/NADPH, while the NADP(H)-dependent dehydrogenase uses the produced cofactor to catalyse the oxidation or reduction of a substrate. By nanoconfining the two catalysts the effective concentration of the enzymes is greatly increased, and diffusion distances of the cofactor minimized. The system makes it possible to drive and monitor complex biotransformations. This Thesis shows that by incorporating the NADPH-dependent Malic Enzyme (ME), the resulting electrode, (FNR+ME)@ITO/Support, is able to drive reductive CO2 incorporation into pyruvate (C3) forming malate (C4). The favourable catalysis within the electrode pores allows for the design of a system which mimics biological oxygenic photosynthesis by incorporating a PEC-PV system with a photoanode in conjunction with a 0.5 V Silicon solar cell. For the first time the Electrochemical Leaf was exploited to drive multi-enzyme cascade reactions where the overall rate is measured directly as an electric current through the FNR-NADPH-dependent dehydrogenase couple. As a proof of concept, the reversible amination of pyruvate (C3) to L-aspartate (C4) was investigated by entrapping FNR, ME, carbonic anhydrase, fumarase and L-aspartate ammonia lyase. The Electrochemical Leaf can be coupled to any natural or artificial multienzyme cascade provided one enzyme is NAD(P)(H) dependent. The implications are numerous for synthesis, biosensing and fundamental investigation of systems catalysis.
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