Periodic Reporting for period 4 - HIGH-GEAR (High-valent protein-coordinated catalytic metal sites: Geometric and Electronic ARchitecture)
Période du rapport: 2021-12-01 au 2023-05-31
Enzymes utilize metals and radicals to perform particularly challenging chemistry in energy conversion, chemical synthesis and for harvesting sunlight. If we could mimic these processes it would eliminate the need for fossil fuels and transform chemical industry while reducing its environmental impact.
The key catalytic species are highly reactive organic radicals or high-valent metal clusters with a varying ligand environment, provided by the protein, that drastically impacts and delicately controls the reactivity. To be able to understand and mimic this chemistry for industrial and medical purposes it is of central importance to know the atomic structure of the cofactor, as well as the entire chemical environment that the protein provides. It has, for a number of reasons, proven extremely challenging to obtain these.
The central goal of this project is to determine high-resolution structures of radical states and high-valent metal site intermediates, coordinated in protein matrices known to direct these for varied and important chemistry. Using new methods for structure determination, advanced spectroscopy, and theory we aim to define atomic-resolution geometric structures of the high-valiant species and describe how the protein controls the cofactor state as well as its chemical reactivity and mechanism for some of the most potent catalysts in nature.
The key catalytic species are highly reactive organic radicals or high-valent metal clusters with a varying ligand environment, provided by the protein, that drastically impacts and delicately controls the reactivity. To be able to understand and mimic this chemistry for industrial and medical purposes it is of central importance to know the atomic structure of the cofactor, as well as the entire chemical environment that the protein provides. It has, for a number of reasons, proven extremely challenging to obtain these.
The central goal of this project is to determine high-resolution structures of radical states and high-valent metal site intermediates, coordinated in protein matrices known to direct these for varied and important chemistry. Using new methods for structure determination, advanced spectroscopy, and theory we aim to define atomic-resolution geometric structures of the high-valiant species and describe how the protein controls the cofactor state as well as its chemical reactivity and mechanism for some of the most potent catalysts in nature.
The first part of the project was dedicated to establishing the protein model systems and methodology. During this time we established reproducible protocols for the production of correctly folded metal free proteins, characterized metal loading schemes and established protocols for micro-crystallization generating samples suitable for XFEL data collection.
Further on in the project, approaching the main objective, we established the drop on tape sample injection/oxygen activation method for our samples and employed it at LCLS in Stanford, SACLA in Japan, and PAL-FEL in South Korea. As a final proof of the advancement and suitability of the methodology and model systems we obtained sub 2Å femtosecond X-ray free-electron laser diffraction datasets of the resting oxidized states of our protein model systems.
During the second half of the project we utilized our model systems and methodology to obtain structures of our metalloproteins in different oxidation states. This provided key information on how the protein influences the metals site and how it is set up to bind substrates as well as control the high-valent catalytic states. We have also been able to characterize redox-induced structural changes in the cofactors and protein matrix and how these are linked to reactivity and control of the chemistry in the cellular context.
Further on in the project, approaching the main objective, we established the drop on tape sample injection/oxygen activation method for our samples and employed it at LCLS in Stanford, SACLA in Japan, and PAL-FEL in South Korea. As a final proof of the advancement and suitability of the methodology and model systems we obtained sub 2Å femtosecond X-ray free-electron laser diffraction datasets of the resting oxidized states of our protein model systems.
During the second half of the project we utilized our model systems and methodology to obtain structures of our metalloproteins in different oxidation states. This provided key information on how the protein influences the metals site and how it is set up to bind substrates as well as control the high-valent catalytic states. We have also been able to characterize redox-induced structural changes in the cofactors and protein matrix and how these are linked to reactivity and control of the chemistry in the cellular context.
The project has yielded a number of important results as described in 15 scientific publications stemming from the studies. For example, we have discovered unusual photochemistry catalyzed by one of our proteins and have further characterized this using crystallography and spectroscopy (J Am Chem Soc. 140(4):1471-1480 (2018)). The characterization of the protein model systems allowed us to identify and describe a new group of aerobic ribonucleotide reductases, present in common pathogens, that function via a novel chemical mechanism (Nature. 563(7731):416-420 (2018)). We have also worked with a new method called micro-crystal electron diffraction (Micro-ED). In our efforts to evaluate if this is suitable to characterize the proteins in this project, we employed this method to one of our proteins. Doing so we managed to solve the first new protein structure using Micro-ED (Science Advances. 5(8):eaax4621 (2019)). We have dissected the protein-metal interplay that guide metal binding and reactivity in our enzymes (J Am Chem Soc. 142:5338-5354 (2020)). We have determined the high resolution XFEL structures of the di-iron Methane Monooxygenase in two oxidation states, showing a full reaction cycle performed at our drop-on-tape oxygen-incubation setup (J Am Chem Soc 142: 14249-14266 (2020)). Finally i would like to specifically mention a study where we were able to characterize redox-controlled structural reorganization within the ribonucleotide reductase R2b-NrdI complex and how that is utilized to control the redox properties of the cofactors in the system (eLife Sep 9;11:e79226 (2022)).