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

Lead Research Organisation: STFC - Laboratories
Department Name: Scientific Computing Department

Abstract

The mechanisms of effective electron and proton transfer in chemical processes, to catalyse chemical reactions and enable essential biochemical functions, are still not fully understood. This proposal combines state of the art experimental and high performance computational methods to address these questions, developing innovative approaches with a focus on proteins that perform fundamental chemistry that is important to the environment as part of the global nitrogen cycle. New experimental methods include rapid room temperature X-ray crystallography and single crystal spectroscopies, while the theoretical approach will be a mixture of quantum mechanics, molecular mechanics and molecular dynamics, with the calculations performed on state of the art parallel processing computer systems.

Copper nitrite reductases are important environmental proteins that carry out the chemistry to convert nitrite (NO2) to nitric oxide (NO) during the process of denitrification, a key step of the biological 'nitrogen cycle' whereby nitrogen gas is returned from the soil to the atmosphere. We will first study this process in an enzyme from Achromobacter cycloclastes. It requires binding of NO2 to a Cu atom and its reduction to NO via mechanisms involving a second Cu atom and electron and proton transfer to the 'active site'. This also normally involves the formation of a normally transient complex between the nitrite reductase and a cytochrome (electron donor) partner protein, making the chemistry difficult to study. Recently a new nitrite reductase has been discovered that contains a 'tethered' cytochrome domain. This protein, from Ralstonia picketti, acts as a 'self-contained electron transfer' system, an unusual and rare structure that negates the need for a transient protein-protein complex and which provides us with the unique opportunity to fully study such a fundamental catalytic process in detail, ie electron transfer, proton transfer, NO2 delivery and binding, as well as the metal oxidation states.

The outcomes of our programme will be of broad relevance to any chemistry involving controlled electron and proton transfer reactions, processes that are ubiquitous in nature, and essential for future development of efficient biomimetic compounds or synthetic enzymes & enzyme-inspired catalysts for industrial applications.

Technical Summary

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.

Planned Impact

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
 
Description This was a joint proposal with awards BB/M022714/1 and BB/M020924/1 to investigate Cu proteins involved in a vital catalytic process in agriculture - the global nitrogen cycle, a process that also has environmental relevance, including formation of greenhouse gases like nitrous oxide. The key findings of our research to date may be summarised by (A) new methods and approaches and (B) structural and electronic insights into catalysis.
A. New Methods and Approaches
(i) Imaging biomolecules during their catalytic cycles was brought a step closer by our work. We developed an approach called 'MSOX' (Multiple Structures from One Crystal) that enabled sequential (serial) atomic resolution structures of nitrite reduction catalysis to be captured at room temperature. From these data, X-ray molecular 'movies' were generated to show the complete catalytic cycle: the resting state enzyme, nitrite binding to the active Cu site, reduction of nitrite to nitric oxide, release of this product and reformation of the resting enzyme. We were successful with a two domain Cu nitrite reductase (CuNiR). However, we were unable to complete planned MSOX experiments on the catalytic cycle of a hybrid haem-Cu protein because nitrite binding to the resting state of this variant was not established. See BB/M022714/1 'Key findings' for further details of the experimental program.
(ii) Increasing the information content and verisimilitude of QM/MM modelling of enzyme catalysis by using hybrid MSOX-QM/MM methods. Typically, QM/MM modelling of an enzyme active site is based on a single static crystal structure, or perhaps the starting and final states as captured in the crystal. We have greatly extended this approach by using the many sequential structures obtained from MSOX experiments to both guide and validate the simulated structures and electronic states of the active site (protein and ligands), and help determine reaction mechanisms. The MSOX-QM/MM simulations of crystal reactions can then act as a reference for simulations of the proteins in solution.
B. Insights in Catalysis
Main specific results to date for Molecular Dynamics, DFT and QM/MM simulations of Cu nitrite reductase active sites can be summarised as follows:
• Protonation states of active site Asp and His residues can 'tune' the water and ligand accessibility of the active site
• The loss of a bound water at the active site in MSOX of the resting enzyme is due to reduction of the active site Cu atom
• In the two domain nitrite-bound enzyme, the initial 'top-hat' orientation of nitrite corresponds to the Cu(II) state. MSOX shows a subsequent 'side-on' orientation and calculations show this is due to reduction of the metal to Cu(I).
• In addition to the side-on intermediate, QM/MM indicates 'reverse side-on' and 'N-bound' nitrite conformations as possible transient states though not observed by MSOX. All these intermediates are capable of forming nitric oxide via similar mechanisms.
• In the haem-Cu hybrid protein, binding of nitrite via either N or O is possible for the Cu(II) state while in the Cu(I) state N-binding is energetically preferred
Exploitation Route Non-Academic Impacts: The experimental PDRA for this joint project, Dr Sam Horrell gained significant training and expertise in advanced structural methodologies and in the use of X-ray beamlines in the UK, France and Switzerland. These skills led to him securing a position at the Center for Free Electron Science, DESY/University of Hamburg where he is currently based. The computational PDRA on the project, Dr Kakali Sen, is currently employed at STFC Hartree Centre, using the skills gained during this project to work on problems related to catalysis in materials for industry.

A number of public engagement activities were undertaken in the scope of the overall joint proposal, including:
-An article aimed at the general public published in 'Research Features' Magazine.
- Public engagement at the large scale STFC Daresbury Laboratory Open Week event.
- Visiting school children engaged with a protein crystallisation activity at Essex.

Other impacts include several workshop presentations at Diamond Light Source describing our research and engaging with the community.
Sectors Chemicals,Environment,Other

 
Title MSOX - Multiple Structures from One Crystal Protein Crystallography 
Description Development of multiple structures from one crystal serial crystallography (MSOX) using synchrotron radiation with rapid data collection pixel detectors and at variable temperatures between 100K and room temperature. 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact A high impact publication in IUCrJ recognised by an editorial overview by leading researchers in the XFEL community where a different serial approach (many crystals-one structure) is used. The MSOX approach shows capability of researchers to use it to obtain (i) radiation damage free structures and (ii) kinetic/dynamic related structural changes in a crystal undergoing catalysis in situ on x-ray source. 
 
Description Collaboration with Prof Rob Thorne, Cornell 
Organisation Cornell University
Department Department of Physics
Country United States 
Sector Academic/University 
PI Contribution In following our currently funded program of research - making crystallographic movies of catalysis at 100K and RT - we saw a need to interrogate the elevated cryogenic regime, above 200K, which is technically difficult. We had crystals and a plan but not the technical experience. Prof Rob Thorne at Cornell had the experience and his team were able to help us perform the necessary experiments. Subsequently, we processed, analysed and interpreted the data collected by the Cornell team. We also linked these data to molecular dynamics and DFT calculations.
Collaborator Contribution Our Cornell collaborator provided us with access to the CHESS synchrotron, beamtime and a team of experimenters for performing the work, using samples (crystals) that we provided for the purpose. These were elevated temperature dependent cryogenic methods (200-280K) not available to us elsewhere. A total of 11 series of MSOX datasets were collected at different temperatures and the data were then provided to us for further work.
Impact A manuscript describing this work ('Active site protein dynamics and solvent accessibility in copper nitrite reductase') using some of the data collected at Cornell, along with extensive computational work conducted in our labs, has been submitted (late Feb 2017) to IUCrJ, with Cornell co-authors.
Start Year 2016
 
Description CCPBioSim Biomolecular QM/MM Modelling with ChemShell workshop, June 2018 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact CCPBioSim held a training day on combined quantum mechanical/molecular mechanical (QM/MM) modelling of biomolecular systems at the University of St Andrews on 14 June 2018 as part of the ScotCHEM 2018 conference. In the morning session we discussed the principles of QM/MM modelling and introduced the ChemShell software package. ChemShell is a scriptable computational chemistry environment which provides a flexible way to link QM and MM codes together to perform QM/MM calculations. There was then an opportunity to learn the basics of ChemShell in the first practical. In the second lecture we described in more depth how QM/MM biomolecular calculations are set up and performed, using a cytochrome P450 system as a case study. The second practical explored modelling of enzymatic reactions with ChemShell on STFC's SCARF cluster.
Year(s) Of Engagement Activity 2018
URL https://www.scotchem.ac.uk/st-andrews-2018/
 
Description CCPBioSim Biomolecular QM/MM Modelling with ChemShell workshop, May 2017 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact CCPBioSim held a training day on combined quantum mechanical/molecular mechanical (QM/MM) modelling of biomolecular systems at STFC Daresbury Laboratory on the 9th May 2017.In the morning session we discussed the principles of QM/MM modelling and introduced the ChemShell software package. ChemShell is a scriptable computational chemistry environment which provides a flexible way to link QM and MM codes together to perform QM/MM calculations. There was then an opportunity to learn the basics of ChemShell in the first practical. In the second lecture we described in more depth how QM/MM biomolecular calculations are set up and performed, using a cytochrome P450 system as a case study. The second practical explored modelling of enzymatic reactions with ChemShell on the Hartree Centre systems. In the final lecture we showcased recent ChemShell developments.
Year(s) Of Engagement Activity 2017
URL http://www.ccpbiosim.ac.uk/events/workshop-course-material/eventdetail/95/-/qm-mm-modelling-of-biomo...
 
Description Cafe Scientifique -a forum for debating science issues 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Cafe Scientifique is a forum for the discussion of current work and interesting scientific issues. They are informal and accessible to people who are interested in science and speakers are there to be questioned and talk about their work at all levels. Sam Horrell presented a talk on 8th March at the Cafe Scientifique in Colchester, Essex on 'The Healing Power of Crystals', focused on structure based drug discovery and our BBSRC funded crystallographic and computational work.
Year(s) Of Engagement Activity 2017
URL http://www.cafescientifique.org/?id=236:colchester
 
Description Daresbury Open Week 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Over 70 activities were available at the Daresbury Laboratory Open Day Week. The purpose was to introduce the public, businesses and schoolchildren to science up close and to show the range of scientific activity at the laboratory. This is where the Hartree Centre is located and we presented a short talk and poster on our BBSRC award related work.
Year(s) Of Engagement Activity 2016
URL http://www.stfc.ac.uk/public-engagement/activities-for-the-public/visit-daresbury-laboratory/daresbu...
 
Description Poster presentation at CCP5 AGM 2016 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Kakali Sen presented a poster on water accessibility and nitrite binding at the T2Cu active site in copper containing nitrite reductases
Year(s) Of Engagement Activity 2016
 
Description Poster presentation at CCP5 Simulation for the Experimentalist and Industrialist workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Chin Yong and Tom Keal presented a poster on water accessibility and nitrite binding at the T2Cu active site in copper containing nitrite reductases
Year(s) Of Engagement Activity 2016
URL https://eventbooking.stfc.ac.uk/news-events/simulations-for-the-experimentalist-and-the-industrialis...