Periodic Reporting for period 3 - Fields4CAT (Force Fields in Redox Enzymatic Catalysis)
Okres sprawozdawczy: 2022-03-01 do 2023-08-31
Working towards a more sustainable society inevitably goes through developing a healthy manufacturing industry that fulfils our societal needs in an economically sustainable and environmentally friendly manner. Among all issues that need to be overcome to get to that aim, a more sustainable industrial chemical synthesis for manufacturing of added value materials, such us drugs, high-specs polymers, etc., needs to be developed. Currently, employed catalytic methods herein often require economically unbearable use of highly expensive catalysts, energetically unfavourable reaction conditions, and use of highly contaminant non-aqueous chemical components. As in many other disciplines, nature teaches us lessons on how to do that in a much more efficient, sustainable way via enzymatic catalysis. Enzymes are able to catalyse key reactions for us at superior rates of several orders of magnitude compared to synthetic paths, and under very mild (ambient) conditions using a fully green aqueous chemistry. Enzymatic catalysis is one of those disciplines in molecular biology where the crystal structure has been unable to bring a definitive mechanistic picture on such an outstanding catalytic process. Knowing the “secrets” of enzymatic catalysis is a major call and a priority in providing sustainability for the future generations. While a number of hypotheses based on pure chemical interactions have attempted to explain enzymatic catalysis in a number of enzyme proteins, this has not yet provided a universal picture on how the reactions are being significantly sped up in an enzyme active site. The Biophysics field has abundantly shown how forces can play key roles in essential biological functions, e.g. the emerging field of mechanobiology shows us how mechanical forces are regulated at the molecular level to control cell functions. A plausible, yet experimentally untested, hypothesis states that nature has also mastered the exploitation of force fields to control chemical reactivity. Fields4CAT focuses on this issue by proposing a unique set of experiments combining single-protein trapping in a nanogap (with full control over applied high force fields) with site-specific protein mutagenesis. This unique approach allows orientation-control trapping of individual enzymes in a nanoscale tunelling gap while continuously monitoring the tunelling current flowing through the protein structure. Abrupt changes in the transient tunnelling current are indicative of enzyme activity (see Summary Figure below) and allow for electrical characterization of enzymatic activity in a single enzyme. The enzymatic activity can be then studied under the application of external force fields stimuli along the nanoscale junction. Revealing the details of how force fields affect enzymatic processes will undoubtedly open to “new ways” of doing more sustainable chemical synthesis by either artificially boosting the enzyme’s enzymatic capabilities or by mimicking the main chemical principles of enzymatic catalysis.
The progress of this ERC project can be summarized as follow:
- We have set up three independent scanning probe-based microscopes with capabilities to create and electrically characterize individual proteins trapped in a nanoscale gap between two conductive electrodes. Among the special features of such equipment, it operates under ambient conditions using a liquid cell to work at (near) physiological conditions, allows temperature and electrochemical control of the cell, and allows the easy implementation of high electric and magnetic force fields along the main nanogap axis. The cell incorporates an optical fiver for local illumination. All setups have been equipped with custom-made current amplifier circuits for high-resolution current measurements and home-made software codes for customized experiments.
- We have characterized the main electrical signatures of relevant co-factors (metalloporphyrins) and biological motifs (alpha-helical peptide sequences), which are the main constituents of the targeted redox enzyme studied in this ERC project. We have made a number of discoveries here: (1) the ability of a supramolecularly trapped metalloporphyrin co-factor to conduct electricity is governed by the chemical details of the interactions leading to molecular trapping in the nanoscale junction. (2) There exist particular electron pathways in the metalloporphyrin moiety promoting spin polarization of the currents. (3) An alpha-helical peptide structure bearing large electrical dipole running through the maim helical axis can control the sign of the spin polarization of the electrons crossing the helical structure.
- We have demonstrated the electrical transduction of enzymatic catalysis in a single enzyme trapped in a nanoscale junction (see Summary for Publication image below). The study allowed the comparison of the slow P450 with a fast Glutathione Reductase enzymes, consolidating the results.
- We have now demonstrated quantum-supported conductivity in a small tetraheme cytochrome protein which links to the observed long-range charge transport observed in multiheme based molecular wires in electrical bacteria. The work demonstrates vi electrochemical control the key role of the redox heme cofactors in catalysing charge transport under a small driving electric field.
- We have progressed on the understanding of the Fe release mechanism in a Ferritin protein under an electrical perturbation. We have identified a passive (non-catalysed) mechanism for the Fe release. We have identified the Fe loading level as a key ingredient in this process and working towards the preparation of Ferritin with very different Fe loadings.
- We have set up three independent scanning probe-based microscopes with capabilities to create and electrically characterize individual proteins trapped in a nanoscale gap between two conductive electrodes. Among the special features of such equipment, it operates under ambient conditions using a liquid cell to work at (near) physiological conditions, allows temperature and electrochemical control of the cell, and allows the easy implementation of high electric and magnetic force fields along the main nanogap axis. The cell incorporates an optical fiver for local illumination. All setups have been equipped with custom-made current amplifier circuits for high-resolution current measurements and home-made software codes for customized experiments.
- We have characterized the main electrical signatures of relevant co-factors (metalloporphyrins) and biological motifs (alpha-helical peptide sequences), which are the main constituents of the targeted redox enzyme studied in this ERC project. We have made a number of discoveries here: (1) the ability of a supramolecularly trapped metalloporphyrin co-factor to conduct electricity is governed by the chemical details of the interactions leading to molecular trapping in the nanoscale junction. (2) There exist particular electron pathways in the metalloporphyrin moiety promoting spin polarization of the currents. (3) An alpha-helical peptide structure bearing large electrical dipole running through the maim helical axis can control the sign of the spin polarization of the electrons crossing the helical structure.
- We have demonstrated the electrical transduction of enzymatic catalysis in a single enzyme trapped in a nanoscale junction (see Summary for Publication image below). The study allowed the comparison of the slow P450 with a fast Glutathione Reductase enzymes, consolidating the results.
- We have now demonstrated quantum-supported conductivity in a small tetraheme cytochrome protein which links to the observed long-range charge transport observed in multiheme based molecular wires in electrical bacteria. The work demonstrates vi electrochemical control the key role of the redox heme cofactors in catalysing charge transport under a small driving electric field.
- We have progressed on the understanding of the Fe release mechanism in a Ferritin protein under an electrical perturbation. We have identified a passive (non-catalysed) mechanism for the Fe release. We have identified the Fe loading level as a key ingredient in this process and working towards the preparation of Ferritin with very different Fe loadings.
Our initial mesurements for testing have brought unprecedented electrical behaviours in molecular moieties which are constituent parts of cytochrome enzymes (Angewandte Chemie, ACS Journals, and media, e.g. ChemistryViews). The main block (WP2) has produced new surface preparation protocols to effectively trap enzymes in a nanoscale junction. Single-protein electrical characterization using our controlled protein trapping in a nanoscale gap has revealed outstanding electrical behaviour in the studied enzymes; (1) we have been able to compare the electrical response of two different enzymes individually trapped in a nanoscale junction and characterize, namely, P450 and Glutathione Reductase, and electrically characterize their distinct enzymatic activity. (2) We have characterized quantum-supported charge transport in a multiheme protein and demonstrated the role of the redox heme cofactor in catalysing this process and leading to the observed long-range charge transport behaviour described in multiheme-based bacterial nanostructures. (3) We have established the most plausible mechanisms for the electrochemically induced Fe release in a Ferritin enzyme, and connect such mechanisms to the Ferritin biological functions as well as the possible avenues for new methods of electrochemical sensing of ferritin in blood serum. Several high-impact publications are now under preparation reporting the above discoveries.
From now until the end of the project, we expect to complete our understanding of the above mechanisms by learning how the above processes respond under magnetic stimuli on top of the applied electric fields. We believe this will help us achieve a complete picture of the effect of forces in the above enzymatic processes involving charge movement.
From now until the end of the project, we expect to complete our understanding of the above mechanisms by learning how the above processes respond under magnetic stimuli on top of the applied electric fields. We believe this will help us achieve a complete picture of the effect of forces in the above enzymatic processes involving charge movement.