Dynamics of Electron and Proton Transfer Chemistry in Copper and Hybrid Copper-Haem Enzymes

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We will study, at the atomistic level using large scale QM/MM and molecular dynamics calculations and rapid room temperature crystallography, the structure, dynamics and mechanisms of copper and haem containing nitrite reductases involved in biological catalysis critical for the global environment. These enzymes carry out difficult chemistry involving controlled electron and proton transfer mechanisms that are ubiquitous in nature and applicable to numerous synthetic chemical and biochemical systems. We have assembled a multidisciplinary team of researchers allowing us to apply a powerful combined computational-experimental approach.

Specifically, we will address the factors that determine the redox properties and electron/proton transfer, substrate (nitrite) and proton delivery pathways of a two-domain copper nitrite reductase (AcNiR) and a novel three-domain, haem-Cu-nitrite reductase tethered 'self-electron transfer' complex (RpNiR). We will use fast-repeat room temperature X-ray crystallography to determine frame-by-frame movies of the dynamics of catalysis and perform high-level hybrid QM/MM modelling and all-atom MD of the Fe-haem, type-1 Cu and type-2 Cu centres and determine the 'minimum enzyme' environment involved in ligand binding, electron and proton transfer and catalysis (nitrite to nitric oxide). The computational and experimental aspects of the project will work synergystically to provide unprecedented dynamic and structural detail for the entire catalytic process. The outcomes will be highly relevant for both academic and industrial applications in mechanistic enzymology and will result in experimental and computational tools for use by the wider UK research communites engaged in developing synthetic enzymes, biomimetics or nanoparticles for catalysis.

Our research is well-aligned to BBSRC strategic priorities, including long-term multidisciplinary research, technology development for biosciences and new tools for chemical biology and high resolution structural analysis, through close engagement with large scale facilities (Hartree & Diamond) and immediate international impact via collaboration with the Swiss Light Source. We are conducting transformative research that provides a paradigm shift, through rapid room temperature crystallography allied to advanced computational chemistry. Each group of beneficiaries is given below along with the means by which they will benefit from the research impact.

Information gained will be relevant to general mechanisms for electron and proton transfer and catalysis in natural and synthetic enzymes, having broad impact for synthetic biology, chemical catalysis, and biotechnology. Thus our work will, in the medium term, be relevant to the UK biotechnology and chemical industries.

- In synthetic biology, we will provide information on 'minimum enzyme' structures required for catalysis
- In chemical catalysis, our contribution will be helpful in the rational design of biomimetic catalysts
- In technology, our work has impact in bioremediation technologies, e.g. the nitrogen cycle, as well as for industrial and academic users of synchrotron facilities.

We will contribute new capabilities to STFC's ChemShell QM/MM package for chemical modelling of native and synthetic enzymes, with benefit to other UK BBSRC researchers, academics and industrial scientists. New software will be placed in the CCPForge repository and included in subsequent releases of the code, freely available to UK academics. Commercial sectors will benefit through inclusion in the Accelrys software 'QMERA'. Experimental developments (e.g. rapid room temperature crystallography) will be made immediately available to the benefit of hundreds of academic and industrial researchers, including major pharma companies and SMEs engaged on chemical catalysis or structure-based drug-discovery programmes. We will seek to further relationships with industry, working closely with Liverpool's Business Gateway and the Research and Enterprise Office at Essex to achieve this aim.

We are committed to wide dissemination of our new methods and best practice, in partnership with the synchrotron laboratories and the Hartree Centre. We believe our project will serve as an exemplar of the great benefits of linking high resolution, room-temperature kinetic crystallography with advanced computational simulation. We aim to inform and inspire other researchers and large scale facility users, industrial and academic, to adopt this approach. Methodologies and outcomes will be publicised by permanent poster displays and at facility User meetings attended by UK academics and industrialists. This will lead to impact in several stakeholder groups, including the chemical industry, enzyme/biocatalysis communities; and academic and industrial groups interested in synthetic biology.

Our proposal offers excellent training for PDRA staff in multidisciplinary science. The PDRAs will develop skill sets of value to both commercial and academic sectors, enhanced by full involvement in international collaboration. The Liverpool PDRA will receive substantial training in highly parallelised high performance computing, addressing the future of the UK's economic development by contributing to a cadre of well trained professional HPC specialists. PDRA secondments to partner sites, including Diamond and Hartree, and extensive use of national and international facilities will maximise the training benefit. This will yield rounded and highly skilled researchers who will be well placed to contribute further to the UK science base and competitiveness of UK industry.

Detailed timelines with milestones for delivery of impact is given in the Workplan and Pathways to Impact