Periodic Reporting for period 4 - Fields4CAT (Force Fields in Redox Enzymatic Catalysis)
Período documentado: 2023-09-01 hasta 2025-02-28
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-controlled 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 so far is summarized below:
- 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.
- We have characterized the main electrical signatures of metalloporphyrins and alpha-helical peptide sequences, which are the main constituents of the redox enzyme studied in this ERC, namely, Cytochrome P450, Small Tetraheme Cytochromes and Ferritin. Main results show: (1) the conductivity of supramolecularly trapped metalloporphyrin is governed by the chemical details of the interactions. (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 shown electrical transduction of enzymatic catalysis in a single enzyme trapped in a nanoscale junction. The study allowed the comparison of the slow P450 with a fast Glutathione Reductase enzymes.
- We have 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 has been now extended to a longer decaheme cytochrome system which corroborates the long-range charge transport model proposed for these protein complexes.
- We have concluded our studies of 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.
- In partnership with ETH Zurick, we have translated the electrostatic catalysis concept exploited by enzymes to two synthetic device platforms; a microfluidic device, and a piezoelectric material’s surface.
- 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.
- We have characterized the main electrical signatures of metalloporphyrins and alpha-helical peptide sequences, which are the main constituents of the redox enzyme studied in this ERC, namely, Cytochrome P450, Small Tetraheme Cytochromes and Ferritin. Main results show: (1) the conductivity of supramolecularly trapped metalloporphyrin is governed by the chemical details of the interactions. (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 shown electrical transduction of enzymatic catalysis in a single enzyme trapped in a nanoscale junction. The study allowed the comparison of the slow P450 with a fast Glutathione Reductase enzymes.
- We have 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 has been now extended to a longer decaheme cytochrome system which corroborates the long-range charge transport model proposed for these protein complexes.
- We have concluded our studies of 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.
- In partnership with ETH Zurick, we have translated the electrostatic catalysis concept exploited by enzymes to two synthetic device platforms; a microfluidic device, and a piezoelectric material’s surface.
Our initial measurements on redox cofactors and alpha-helical peptides (WP1) have brought unprecedented electrical behaviours in these molecular moieties which are constituent parts of cytochrome enzymes (Angewandte Chemie, ACS, and 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, namely, P450 and Glutathione Reductase, and electrically characterize their distinct enzymatic activity at the single-enzyme level. (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 long-range charge transport behaviour. (3) We have established 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. (4) We have demonstrated biology-inspired electrostatic catalysis in two different technological platforms, namely, a microfluidic channel and a piezoelectric material. The work has generated a number of high-impact publications (JACS, Nature Communications, Chemical Science).
From now until the end of the project, the focus will be on the last aim (WP3, section 3.2) which centres around the effect of forcefields in the enzymatic process. We have characterised charge transport in peptide structures and in the metalloenzymes under electron spin polarization, which will reveal untapped mechanistic aspects of enzymatic catalysis under magnetic forcefields. In the last annuity of the project, we foresee achieving a clear picture of the single-enzyme turnover activity behaves under magnetically polarised electric currents.
From now until the end of the project, the focus will be on the last aim (WP3, section 3.2) which centres around the effect of forcefields in the enzymatic process. We have characterised charge transport in peptide structures and in the metalloenzymes under electron spin polarization, which will reveal untapped mechanistic aspects of enzymatic catalysis under magnetic forcefields. In the last annuity of the project, we foresee achieving a clear picture of the single-enzyme turnover activity behaves under magnetically polarised electric currents.