Development of a microscopic gas diffusion-reaction model for a H2 producing biocatalyst

Lead Research Organisation: University College London
Department Name: Physics and Astronomy

Abstract

Providing the technology for production of renewable energy is one of the grand
challenges of this century. There are alternatives to oil, gas and nuclear such as
water, wind and solar power. Of those, the latter is a virtually unlimited power source
and we think that every effort should be undertaken to try to harvest the power
of the sun. This is not an easy task because light energy needs to
be converted into a form of energy that can be stored and supplied
on demand. A convenient storage medium are molecules comprised of atoms
that are held together by energy-rich covalent bonds. Indeed, over millions of years
nature has stored sun light in form of organic molecules (fossil fuels) via natural
photosynthesis. A carbon-free alternative storage medium is molecular hydrogen with
the added advantage that the energy density that can be stored with hydrogen
is significantly larger than for fossil fuels. Thus, molecular hydrogen is envisaged as one of
the primary energy carriers of the future. One of the grand challenges for scientists
is to find or design a cheap catalyst that allows for efficient production of hydrogen from
sunlight and a source for hydrogen atoms, ideally water.

Clearly, one of the most sustainable approaches to hydrogen production is
photocatalytic water oxidation, although this process requires efficient catalysts.
Their design is by no means trivial and can probably be considered as the holy grail
of contemporary material science. A viable alternative that we investigate here
is to exploit biological molecules (hydrogenases) that can be found in microbes
such as green algae and cyanobacteria capable of photosynthetic water splitting.
Pilot plants of H2 producing organisms exist, but there are major barriers that must
be overcome to bring the process to commercial viability. The most important one that
needs to be addressed is the high sensitivity of the organism's hydrogenase to
molecular oxygen. Evolved under anaerobic conditions, the biomolecule gets inhibited or
damaged upon exposure of the oxygen that is around us in the atmosphere.

There is evidence that hydrogenases may be modified so as to render the molecule
less sensitive to oxygen. In order to facilitate this optimization process we propose
here to investigate theoretically the primary events of the oxidative damage, that is diffusion
and binding of oxygen molecules to the active site of hydrogenases, by developing
novel molecular simulation methods. The simulations will help to understand and
interpret recent experimental measurements on a molecular level. For example,
they will allow us to understand which pathways oxygen molecules take before they
damage the active site and how fast this process occurs. The microscopic information
gained from simulation will be vital for the suggestion of modifications (mutations)
of hydrogenase that aim to restrict the access and the binding of molecular oxygen
while leaving the catalytic power for hydrogen production unchanged. The effects
of the suggested mutations will be predicted by our simulations and tested in vitro
by an experimental colleague.

The long term goal of this project is to obtain a hydrogenase mutant with
significantly increased aerotolerance, which can be used for hydrogen production
on a technological scale. This would have a tremendous socio-economic impact
as the hydrogen industry is likely to take a prominent position on the
future energy market.

Planned Impact

The results of the research proposed will reach out to a diverse range of people and
communities including

- academics interested in fundamental and applied bioenergy research detailed in the section
'Academic Beneficiaries'.

- Users of the popular Gromacs computer simulation program, benefitting from code development
as proposed in the computational programme.

- Start-up or spin-off companies in the emerging area of biohydrogen production

- Those in the alternative energy sector with interests in exploiting microbes or hydrogenase
in biofuel cell designs

These beneficiaries will be alerted to our findings by their timely presentation at conferences, in publications and through press-releases timed to coincide with the publication of our research in leading journals. They will also gain from access to the computational and biological resources generated during the project. We anticipate that this impact will begin to be realised from month 18 of the proposed programme of research through the activities of all of the research team. To highlight our research and its potential impact to these groups we will invite representatives to a
workshop on this topic in the second half of the grant period. This conference will bring together eminent
speakers from academia and industry selected for their leading, international reputation in the area.

The work proposed will also shape the personal development of the two PDRAs. They will gain skills in
advancing computational methods and electronic structure theory under the guidance of the PIs. In addition,
the synergistic nature of the research programme and regular meetings of the research team will ensure that the PDRAs gain an understanding of the complementary approaches being used. This together with the multi-site nature of the project will ensure the PDRAs improve their skills in working collaboratively, and communicating effectively within and across sites. They will gain experience of project management under the guidance of the PIs who will also mentor their skills in oral, written and web-based communication of their findings. These impacts will begin at the outset of the project and continue to its completion.

Publications

10 25 50
 
Description (1) We have developed a reaction diffusion model for binding of small ligands to buried enzyme active sites. An application has been carried out on CO-dehydrogenase where we carried out the simulation of CO and CO2 diffusion to the protein active site. We discovered new gas diffusion
channels for CO2 diffusing to the active site and we could explain the directionality for CO diffusion between active sites in the protein
J. Am. Chem. Soc., vol. 135, p. 9493, 2013. We have also developed a sensitivity analysis method, finding the protein residues that when
mutated will give the largest change on the ligand binding rate. J. Chem. Theory Comput., vol. 11, p. 1919, 2015.

(2) We have carried out MD simulations for CO diffusion in FeFe Hydrogenase, which, together with De Gioia's QM calculations (Milan), could be used to interpret Leger's electrochemical measurements on oxidative inhibition of the enzyme. Nature Chemistry, vol. 6, p. 336, 2014.

(3) Following our computational investigation of O2 binding to Fe-Fe hydrogenase (Angew. Chem. Int. Ed., vol. 53, pp. 4081-4084, 2014. ), we have made suggestions for mutations of the enzyme that could reduce the binding affinity of O2. These suggestions were based on the observation obtained from calculations that, following O2 binding, an electron transfers from the catalytic H-cluster site to O2, forming a partially oxidized cubane and O2-. The aim was to counteract this electron transfer by placing positively charge residues close to the cubane cluster and negatively charged residues close to the O2 binding site. Following mutations were suggested to the group of Leger: Thr356Lys, Ile356Lys, Cys298Glu. Unfortunately, these mutations did either not yield a stable enzyme or did not show reduction in oxygen binding.

(4) By combining electrochemistry, site-directed mutagenesis, molecular dynamics and quantum chemical calculations we uncovered the molecular mechanism of O2 diffusion within the FeFe hydrogenase enzyme and its reactions at the active site. We find that the partial reversibility of the reaction with O2 results from the four-electron reduction of O2 to water. The third electron/proton transfer step is the bottleneck for water production, competing with formation of a highly reactive OH radical and hydroxylated cysteine. The rapid delivery of electrons and protons to the active site is therefore crucial to prevent the accumulation of these aggressive species during prolonged O2 exposure. These findings should provide important new clues for the design of hydrogenase mutants with increased resistance to oxidative damage.
Exploitation Route Two lines of investigation are worth pursuing in future work to reduce oxygen sensitivity of FeFe hydrogenases.
The first is the screening of the effects of all possible substitutions at positions 169, 290 and 296 (Cr numbering)
of using both electrochemistry and MD simulation, with the aim to restrict O2 access to the active site and slow
the formation of the dead-end species. The second is the systematic search for mutations that accelerate the
third ET/PT step of O2 reduction at the active site using electrochemistry and QM calculations, to eliminate likely
bottlenecks for O2 reduction.
Sectors Chemicals,Energy,Environment

 
Description A key advance during the grant period was the development of a general reaction-diffusion model for ligand binding to enzymes as well as a sensitivity analysis tool identifying protein "hot-spots" that when mutated have the greatest affect on ligand binding. We are confident that these novel approaches will be taken up not only by academic resarchers but also by industrial researchers in the future. The project has resulted in 7 peer-reviewed publications, four of which in the top chemistry journals (Nature Chemistry 2x, Angewandte and JACS). We have been invited to write a review article on our work in an excellent review journal (Methods in Enzymology). The grant facilitated our collaboration with the experimental group of C Leger (CNRS Marseille) and the computational group of L De Gioia (Milan) and made our activities visible to UK and overseas researchers. The project has contributed to the personal development of the PDRAs (A Kubas, D De Sancho). The successful outcome of our project helped Kubas obtain a permanent job in his home country, at the Polish Academy of Sciences where is about to start his own research group. Similarly, De Sancho obtained a personal research fellowship in his home country. This grant has also helped to disseminate and advertise the research of my group to a wider audience and strengthened my case for promotion to Professor.
First Year Of Impact 2014
Sector Chemicals,Energy,Environment
Impact Types Cultural

 
Description Additional funding for international conference on the project theme: "Interface between Experimental and Theoretical Approaches to Energy-related Enzyme Catalysis", June 6-8 2014, UCL, http://www.thomasyoungcentre.org/enzyme-catalysis/
Amount $2,000 (USD)
Organisation Society for Biological Inorganic Chemistry 
Sector Charity/Non Profit
Country United States
Start 06/2014 
End 06/2014
 
Description Additional funding for international conference on the project theme: "Interface between Experimental and Theoretical Approaches to Energy-related Enzyme Catalysis", June 6-8 2014, UCL, http://www.thomasyoungcentre.org/enzyme-catalysis/
Amount £2,000 (GBP)
Organisation Royal Society of Chemistry 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2014 
End 06/2014
 
Description Additional funding for international conference on the project theme: "Interface between Experimental and Theoretical Approaches to Energy-related Enzyme Catalysis", June 6-8 2014, UCL, http://www.thomasyoungcentre.org/enzyme-catalysis/
Amount $5,000 (USD)
Organisation US Navy 
Department US Office of Naval Research Global
Sector Academic/University
Country United States
Start 06/2014 
End 06/2014
 
Description Combining experimental and theoretical methods to learn about the reactivity of gas-processing metalloenzymes 
Organisation National Center for Scientific Research (Centre National de la Recherche Scientifique CNRS)
Department Centre National de la Recherche Scientifique Marseille
Country France 
Sector Academic/University 
PI Contribution (1) We have extended the reaction diffusion model to include the chemical binding step of ligands to enzyme active sites. An application has been carried out on CO-dehydrogenase where we carried out the simulation of CO and CO2 diffusion in the protein. See Ref 1. in box "Output" (2) We have carried out MD simulations for CO diffusion in FeFe Hydrogenase, which, together with De Gioia's QM calculations (Milan), could be used to interpret Leger's electrochemical measurements on oxidative inhibition of the enzyme. See Ref 2 in box "Output". (3) We and de Gioia's group have visited Leger's group in Marseille for two weeks in 2014 (funded by IMERA). This research stay resulted in a review on modern experimental and theoretical methods for investigation of the reactivity of gas-processing metalloenzymes. See Ref 3 in box "Output". (4) Following our computational investigation of O2 binding to Fe-Fe hydrogenase (Kubas et al Angewandte Chemie 2014), we have made suggestions for mutations of the enzyme that could reduce the binding affinity of O2 (specific aim III of the proposal). These suggestions were based on the observation obtained from calculations that, following O2 binding, an electron transfers from the catalytic H-cluster site to O2, forming a partially oxidized cubane and O2-. The aim was to counteract this electron transfer by placing positively charge residues close to the cubane cluster and negatively charged residues close to the O2 binding site. Following mutations were suggested to the group of Leger: Thr356->Lys, Ile356->Lys, Cys298->Glu (5) Following our simulation of O2 diffusion in FeFe hydrogenase, we suggested the following mutations to slow O2 diffusion into active site: Cr V296F, Cr F290Y, Cr F290W.
Collaborator Contribution (1) The group of L de Gioia (Milan) has carried out QM calculations for ligand binding to CO-dehydrogenase. See Ref.1 in box "Output" (2) de Gioia has carried out QM calculations and Leger (CNRS) has carried out electrochemical measurements on FeFe Hydrogenase. See Ref.2 in box "Output" (3) See (3) in box above (4) Leger's group and collaborators carried out the mutations as suggested above 4). While the enzymes were stable, the O2 inhibition rate was not significantly affected by these mutations. We found that no other mutation around the active site was feasible as most of these residues are conserved or have a structural role. Due to the limited success, the mutations studies were not published. (5) Leger's group carried out the mutations as suggested above 5). Mutations Cr V296F, Cr F290Y lead to a significant slowing of O2 diffusion rate (factor 10), whereas the Cr F290W mutant was inactive.
Impact [1] P. Wang, M. Bruschi, L. De Gioia, J. Blumberger "Uncovering a dynamically formed substrate access tunnel in carbon monoxide dehydrogenase/acetyl-CoA synthase", J. Am. Chem. Soc. 135, 9493 (2013). [2] V. Fourmond, C. Greco, K. Sybirna, C. Baffert, P. Wang, P. Ezanno, M. Montefiori, M. Bruschi, I. Meynial-Salles, P. Soucaille, J. Blumberger, H. Bottin, L. De Gioia, C. Leger "The oxidative inactivation of FeFe hydrogenase reveals the plasticity of the H-cluster", Nature Chemistry 6, 336 (2014). [3] C. Greco, V. Fourmond, C. Baffert, P. Wang, P. Bertrand, M. Bruschi, J. Blumberger, L. De Gioia, C. Leger "Combining experimental and theoretical methods to learn about the reactivity of gas-processing metalloenzymes ", Energy Environ. Sci. 7, 3543 (2014). [4] A. Kubas, C. Orain, D. De Sancho, L. Saujet, M. Sensi, C. Gauquelin, I. Meynial-Salles, P. Soucaille, H. Bottin, C. Baffert, V. Fourmond, R. B. Best, J. Blumberger, and C. Leger, "Mechanism of O2 diffusion and reduction in FeFe hydrogenase" Nature Chemistry, vol. 9, pp. 88-95, 2017.
Start Year 2012
 
Description Combining experimental and theoretical methods to learn about the reactivity of gas-processing metalloenzymes 
Organisation University of Milano-Bicocca
Country Italy 
Sector Academic/University 
PI Contribution (1) We have extended the reaction diffusion model to include the chemical binding step of ligands to enzyme active sites. An application has been carried out on CO-dehydrogenase where we carried out the simulation of CO and CO2 diffusion in the protein. See Ref 1. in box "Output" (2) We have carried out MD simulations for CO diffusion in FeFe Hydrogenase, which, together with De Gioia's QM calculations (Milan), could be used to interpret Leger's electrochemical measurements on oxidative inhibition of the enzyme. See Ref 2 in box "Output". (3) We and de Gioia's group have visited Leger's group in Marseille for two weeks in 2014 (funded by IMERA). This research stay resulted in a review on modern experimental and theoretical methods for investigation of the reactivity of gas-processing metalloenzymes. See Ref 3 in box "Output". (4) Following our computational investigation of O2 binding to Fe-Fe hydrogenase (Kubas et al Angewandte Chemie 2014), we have made suggestions for mutations of the enzyme that could reduce the binding affinity of O2 (specific aim III of the proposal). These suggestions were based on the observation obtained from calculations that, following O2 binding, an electron transfers from the catalytic H-cluster site to O2, forming a partially oxidized cubane and O2-. The aim was to counteract this electron transfer by placing positively charge residues close to the cubane cluster and negatively charged residues close to the O2 binding site. Following mutations were suggested to the group of Leger: Thr356->Lys, Ile356->Lys, Cys298->Glu (5) Following our simulation of O2 diffusion in FeFe hydrogenase, we suggested the following mutations to slow O2 diffusion into active site: Cr V296F, Cr F290Y, Cr F290W.
Collaborator Contribution (1) The group of L de Gioia (Milan) has carried out QM calculations for ligand binding to CO-dehydrogenase. See Ref.1 in box "Output" (2) de Gioia has carried out QM calculations and Leger (CNRS) has carried out electrochemical measurements on FeFe Hydrogenase. See Ref.2 in box "Output" (3) See (3) in box above (4) Leger's group and collaborators carried out the mutations as suggested above 4). While the enzymes were stable, the O2 inhibition rate was not significantly affected by these mutations. We found that no other mutation around the active site was feasible as most of these residues are conserved or have a structural role. Due to the limited success, the mutations studies were not published. (5) Leger's group carried out the mutations as suggested above 5). Mutations Cr V296F, Cr F290Y lead to a significant slowing of O2 diffusion rate (factor 10), whereas the Cr F290W mutant was inactive.
Impact [1] P. Wang, M. Bruschi, L. De Gioia, J. Blumberger "Uncovering a dynamically formed substrate access tunnel in carbon monoxide dehydrogenase/acetyl-CoA synthase", J. Am. Chem. Soc. 135, 9493 (2013). [2] V. Fourmond, C. Greco, K. Sybirna, C. Baffert, P. Wang, P. Ezanno, M. Montefiori, M. Bruschi, I. Meynial-Salles, P. Soucaille, J. Blumberger, H. Bottin, L. De Gioia, C. Leger "The oxidative inactivation of FeFe hydrogenase reveals the plasticity of the H-cluster", Nature Chemistry 6, 336 (2014). [3] C. Greco, V. Fourmond, C. Baffert, P. Wang, P. Bertrand, M. Bruschi, J. Blumberger, L. De Gioia, C. Leger "Combining experimental and theoretical methods to learn about the reactivity of gas-processing metalloenzymes ", Energy Environ. Sci. 7, 3543 (2014). [4] A. Kubas, C. Orain, D. De Sancho, L. Saujet, M. Sensi, C. Gauquelin, I. Meynial-Salles, P. Soucaille, H. Bottin, C. Baffert, V. Fourmond, R. B. Best, J. Blumberger, and C. Leger, "Mechanism of O2 diffusion and reduction in FeFe hydrogenase" Nature Chemistry, vol. 9, pp. 88-95, 2017.
Start Year 2012
 
Description Invited Tutorial Lecture 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact "Molecular dynamics simulation of electron transport in proteins", Tutorial on ``Theoretical
Methods for the Functional Analysis of Inorganic Complexes and Metalloenzymes", IMERA,
Marseille, France.

The talk contributed to a better understanding of our computational methods among the group members and collaborators
of C Leger (CNRS Marseille)
Year(s) Of Engagement Activity 2014