Periodic Reporting for period 4 - BiocatSusChem (Biocatalysis for Sustainable Chemistry – Understanding Oxidation/Reduction of Small Molecules by Redox Metalloenzymes via a Suite of Steady State and Transient Infrared Electrochemical Methods)
Reporting period: 2023-09-01 to 2025-02-28
This project addressed significant global challenges in catalysis for energy and sustainable chemistry via learning from, and exploiting, nature’s enzyme catalysts. Metal-containing enzymes within microorganisms catalyse the transformation of carbon dioxide into simple carbon building blocks or fuels, the reduction of dinitrogen to ammonia under ambient conditions and the production and utilisation of dihydrogen. Catalytic sites for these reactions within the enzymes are necessarily based on metals that are abundant in the environment, including iron, nickel and molybdenum. However, attempts to generate biomimetic catalysts have largely failed to reproduce the high activity, stability and selectivity of enzymes. One important key to the exquisite catalysis of enzymes is the choreography of proton and electron transfer and substrate binding during catalytic turnover, and this project set out to establish and utilise a suite of spectroscopic and structural techniques to understand proton-coupled electron transfer and how this is timed with substrate binding and transformation in enzymes.
Key objectives of the action were (i) to establish operando techniques which can be used to understand the intricately controlled chemistry of enzymes; (ii) to apply these to hydrogenases to see what catalytic lessons we can glean from understanding how hydrogen gas is activated cleanly in biology, and how we can exploit hydrogenases themselves in cleaner chemical production; (iii) to apply similar approaches to nitrogenase to understand how natural nitrogen fixation to ammonia can be conducted under ambient conditions in contrast to the high temperature/pressure Haber Bosch process; (iv) to understand the selectivity and efficiency of biological CO2 reduction; and (v) to disseminate these lessons to the wider catalysis community to contribution new catalyst design principles.
The project successfully established operando techniques which will be valuable across structural enzymology, and which have revealed steps in metalloenzyme catalysis. Using a combination of electrochemistry to poise protein crystals in defined redox states, and infrared microspectroscopy and EPR spectroscopy to verify redox state, we were able to record x-ray diffraction structures of metalloenzymes including hydrogenase and nitrogenase in redox states relevant to catalysis. We have gained insight into proton-coupled electron transfer steps, such as the role of subtle movement in a glutamate side change in the mechanism of NiFe hydrogenase. These findings contribute new insight for the design of catalysts for energy technologies for clean, sustainable fuel production and fuel cells, and more energy-economical industrial chemical processes. We have also discovered new ways of harnessing hydrogenase enzymes for hydrogenation reactions under mild conditions, including the hydrogenation of nitro-compounds, and of flavins for coupling to various biocatalytic reductions. This opens up cleaner, simpler biocatalysis for chemical manufacturing, particularly for pharmaceuticals and other fine chemicals. We have disseminated these findings to the research community via a wide array of conference talks and publications. Overall, the findings of the project benefit society by contributing to the urgent challenges posed by global climate change and energy/climate inequality, including cleaner chemical manufacturing, and the need for catalysis for hydrogen cycling and ammonia synthesis.
Key objectives of the action were (i) to establish operando techniques which can be used to understand the intricately controlled chemistry of enzymes; (ii) to apply these to hydrogenases to see what catalytic lessons we can glean from understanding how hydrogen gas is activated cleanly in biology, and how we can exploit hydrogenases themselves in cleaner chemical production; (iii) to apply similar approaches to nitrogenase to understand how natural nitrogen fixation to ammonia can be conducted under ambient conditions in contrast to the high temperature/pressure Haber Bosch process; (iv) to understand the selectivity and efficiency of biological CO2 reduction; and (v) to disseminate these lessons to the wider catalysis community to contribution new catalyst design principles.
The project successfully established operando techniques which will be valuable across structural enzymology, and which have revealed steps in metalloenzyme catalysis. Using a combination of electrochemistry to poise protein crystals in defined redox states, and infrared microspectroscopy and EPR spectroscopy to verify redox state, we were able to record x-ray diffraction structures of metalloenzymes including hydrogenase and nitrogenase in redox states relevant to catalysis. We have gained insight into proton-coupled electron transfer steps, such as the role of subtle movement in a glutamate side change in the mechanism of NiFe hydrogenase. These findings contribute new insight for the design of catalysts for energy technologies for clean, sustainable fuel production and fuel cells, and more energy-economical industrial chemical processes. We have also discovered new ways of harnessing hydrogenase enzymes for hydrogenation reactions under mild conditions, including the hydrogenation of nitro-compounds, and of flavins for coupling to various biocatalytic reductions. This opens up cleaner, simpler biocatalysis for chemical manufacturing, particularly for pharmaceuticals and other fine chemicals. We have disseminated these findings to the research community via a wide array of conference talks and publications. Overall, the findings of the project benefit society by contributing to the urgent challenges posed by global climate change and energy/climate inequality, including cleaner chemical manufacturing, and the need for catalysis for hydrogen cycling and ammonia synthesis.
Overall during this project:
* We have established routes to poising protein crystals in defined redox states, using spectroscopy to verify these redox states, and retrieving crystals for high resolution structural analysis.
* This has enabled structures of hydrogenase in redox states relevant to catalysis, evaluation of proton transfer pathways, and evaluation of redox-linked structure-change in nitrogenase.
*We have further extended our electrochemical crystal-poising methodology to carbon monoxide dehydrogenase and ferredoxins
* We have established the use of cyanophenylalanine as a means to probe redox state of iron sulfur clusters in metalloenzymes, including hydrogenase.
*We have developed ways to harness hydrogenases in hydrogenation of organic compounds, including nitroarenes, which will be valuable in more sustainable chemical manufacturing. Two patents have been filed, and licenced to industry for commercialisation in cleaner chemical manufacturing.
*The project results have been dissemination by 20+ publications (with more in peer review and soon to be submitted, as well as Plenary, Keynote and Invited lectures and conferences across fields of heterogeneous and homogeneous catalysis, biological chemistry, industrial biotechnology, structural biology, metals in biology, and inorganic chemistry.
* We have established routes to poising protein crystals in defined redox states, using spectroscopy to verify these redox states, and retrieving crystals for high resolution structural analysis.
* This has enabled structures of hydrogenase in redox states relevant to catalysis, evaluation of proton transfer pathways, and evaluation of redox-linked structure-change in nitrogenase.
*We have further extended our electrochemical crystal-poising methodology to carbon monoxide dehydrogenase and ferredoxins
* We have established the use of cyanophenylalanine as a means to probe redox state of iron sulfur clusters in metalloenzymes, including hydrogenase.
*We have developed ways to harness hydrogenases in hydrogenation of organic compounds, including nitroarenes, which will be valuable in more sustainable chemical manufacturing. Two patents have been filed, and licenced to industry for commercialisation in cleaner chemical manufacturing.
*The project results have been dissemination by 20+ publications (with more in peer review and soon to be submitted, as well as Plenary, Keynote and Invited lectures and conferences across fields of heterogeneous and homogeneous catalysis, biological chemistry, industrial biotechnology, structural biology, metals in biology, and inorganic chemistry.
* The project has developed electrochemical control methods that allow protein crystals to be poised to precise redox states relevant to catalysis. These go significantly beyond state-of-the-art because previously it has been difficult to obtain crystals of complex metalloenzymes in well-defined, pure redox states. This opens up many new opportunities in structural enzymology.
* We have already used these methods to go beyond state-of-the-art in mechanistic understanding of hydrogenases and nitrogenase, revealing steps in redox-triggered proton transfer in hydrogenase and structural change in nitrogenase.
* The sample cell we developed for in situ electrochemistry-IR microspectroscopy has been provided to MIRIAM beamline at Diamond Light Source (UK synchrotron) for other users in application areas as diverse as heterogeneous CO2 reduction catalysis and gas uptake into MOFs.
* We have gone significantly beyond state-of-the-art in applications of hydrogenases, showing that hydrogenase on a carbon support is a highly effective catalyst for hydrogenation of nitro-compounds to generate amines under a mild conditions. The prevalence of amines in pharmaceuticals and other fine chemicals gives this broad industrial relevance. We have also shown a range of applications of hydrogenase-driven flavin recycling in innovative areas of biocatalysis for more sustainable chemical manufacturing. Prompt patent protection and licencing to an SME company has provided an efficient route for translation to industrial application.
* We have already used these methods to go beyond state-of-the-art in mechanistic understanding of hydrogenases and nitrogenase, revealing steps in redox-triggered proton transfer in hydrogenase and structural change in nitrogenase.
* The sample cell we developed for in situ electrochemistry-IR microspectroscopy has been provided to MIRIAM beamline at Diamond Light Source (UK synchrotron) for other users in application areas as diverse as heterogeneous CO2 reduction catalysis and gas uptake into MOFs.
* We have gone significantly beyond state-of-the-art in applications of hydrogenases, showing that hydrogenase on a carbon support is a highly effective catalyst for hydrogenation of nitro-compounds to generate amines under a mild conditions. The prevalence of amines in pharmaceuticals and other fine chemicals gives this broad industrial relevance. We have also shown a range of applications of hydrogenase-driven flavin recycling in innovative areas of biocatalysis for more sustainable chemical manufacturing. Prompt patent protection and licencing to an SME company has provided an efficient route for translation to industrial application.