Eagle Eye Magazine Issue 1 2023

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EAGLE EYE | ISSUE 1 | <strong>2023</strong> Figure 1: Huc hydrogenase - The large subunit and small subunit are shown in lime and orange respectively. Magnesium is shown as a magenta sphere. The iron-sulphur clusters, which transfer electrons to and from the active site, are in translucent yellow, while the active site itself is in a translucent blue-pink gradient. Figure 2: High occupancy volumes, representing areas where A) oxygen and B) hydrogen is present > 4% of the time during the simulations. Two major cavities were found within the enzyme which are significantly involved in gas transportation, show in red and blue respectively. time was the second best ever achieved on any protein by CryoEM, confidently allowing the structure-to-function relationship of the hydrogenase to be determined for the first time, which is where the computational protein dynamics work I did began. Your research focused on molecular modelling and simulations. How did this work fit into the overall research project? My work involved determining how the structure of the hydrogenase could allow efficient discrimination between hydrogen and oxygen, despite their similarity. This involved establishing the different tunnels and pathways which allowed the gases to diffuse closer to the reaction site. Along these paths, I witnessed differences in how oxygen and hydrogen moved between areas of the protein. The diffusion pathway in Huc traverses multiple bottlenecks and other points of selection where hydrogen, with weaker non-polar interactions and a slightly smaller radius, can diffuse far more easily than the bulkier oxygen. Most of the identified bottlenecks were found to be caused by large branched apolar residues, simply discriminating according to size. These specific residues have been identified in other hydrogenase homologues with increased oxygen, slowing the rate that oxygen accesses the active site compared to hydrogen. However, since proteins are dynamic, the channel changes shape and size over time allowing some oxygen through, which is why hydrogenase up until this point has retained oxygen sensitivity. Crucially, I identified a selection point close to the active site which was 100% efficient at occluding oxygen in all my simulations, which hasn’t been seen in any other hydrogenases. I then studied the effect of various mutations on the selectivity of hydrogenase, to help determine which residues specifically within Huc hydrogenase are responsible. My further research, after submission of the paper, suggests that a nearby residue at this site moves in response to the quadrupole of the oxygen molecule, occluding the channel to prevent access to the active site only when oxygen is present. This work has confirmed that the effects of selection are due to specific mutations with the gas channels, which was suggested by the group and has determined residues likely responsible for selection, aiding further research in vivo and in silico to create hydrogenases for cleaner, accessible energy sources. What was most challenging about this research? Within molecular dynamics, one of the hardest hurdles to overcome is the interaction of metal ions with proteins. Classical molecular dynamics utilises simple Newtonian mechanics to simulate the interactions between atoms. This essentially defines the interactions within a system, including steric restraints like distance and angles like a series of balls and springs, applying greater force the further they are from an equilibrium position, while also accounting for non-polar and charge-based interactions. This method uses the position of all atoms in a system with respect to each other to calculate a net force based on a set of parameters which we help define. The issue is that while this classical interpretation works well for most atoms, the interactions between metal ions and protein residues are far more complex, greatly relying on complex quantum interactions involving the overlap of electron orbitals. Defining quantum behaviour through purely classical means to even a moderate level of accuracy is extremely difficult, yet actually calculating these interactions would be far too computationally expensive. For most simulations, the force on each atom is normally calculated for every one 500,000 th of a nanosecond, meaning that the time the supercomputer would take to calculate simulations of the length required for studying hydrogenase dynamics in a partially quantum system would likely 24
far exceed the short timespan I had for this project. Thus, I had to compile a purely Newtonian set of parameters which could reliably reproduce similar dynamics for each of the four metal sites within Huc, which took hundreds of attempts. The breakthrough discovery has been described as a ‘natural battery’. Although it’s early days, can you describe how the enzyme could be used in applications that support a more sustainable future? The potential green applications of an efficient oxygen tolerant hydrogenase are widespread. Sustainable electricity is, of course, key among these: recently it has been discovered that aerobic bacteria such as M. smegmatis are responsible for oxidising approximately 60 billion tonnes of hydrogen per year, which is a huge untapped energy source. Existing hydrogenase biofuel cells use hydrogenases to produce electrons at an anode and couple this to an oxygen-reducing oxidoreductase at a cathode to create an electric current fueled only by the reaction of molecular hydrogen and oxygen into water, producing no greenhouse gases or any other pollutants. These systems have been highly constrained previously, since the entire cathode would have to be placed in protected anaerobic conditions and would require hydrogen to be pumped in manually from sources which would carry some level of environmental cost. However, with Huc hydrogenase, and similar variants immune to oxygen inhibition, this issue will hopefully no longer exist. Eventually, oxygen-tolerant hydrogenases could be used to create cells which can be exposed to aerobic conditions and source hydrogen directly from the atmosphere alone. This hydrogen is pre-existing, produced from fermentation and other natural processes happening across the globe. It is theorised that hydrogen functions indirectly as a greenhouse gas, thus fuel cells removing this from the atmosphere is potentially beneficial. Of course, this is not the only way in which these hydrogenases can be used towards sustainability. In another example, modern enzyme technology allows the production of medicines and other bioactive compounds via eco-friendly methods. However, enzymes containing cofactors like NAD(H) and NADP(H) are required for certain redox reactions. These cofactors and their enzymes are expensive, and their ability to regenerate is key to making sure that companies afford to use them rather than turn to other methods which involve cheaper unsustainable reagents with high carbon footprints and toxic by-products. Luckily, hydrogenases can regenerate these cofactors relatively easily and as a result of creating hydrogenases which require fewer controlled conditions, less specialist equipment and no external fuel sources, this incentivises companies to use these more environmentallyfriendly methods. What are you working on now? My current work is looking into antibiotic resistance, specifically towards the last resort drugs called polymyxins. These drugs are normally only used when all other treatments have failed, but increasing use in recent years has resulted in resistance to even these treatments. Polymyxins are positively charged lipopeptides which are proposed to kill bacteria via interacting electrostatically with negatively charged lipid A, an endotoxin located in gram-negative bacterial membranes, eventually leading to membrane rupture. To combat this, many strains of multidrug resistant bacteria, including nosocomial Salmonella, Pseudomonas and Staphylococcus lineages, modify lipid A with specific chemical groups, like positively charged sugars, which prevent the membrane from being targeted by polymyxins via charge-charge interactions. This project extends upon the techniques used during my masters, creating computational structures of known Lipid A modifying metalloenzymes, including MCR-1 and recently resolved ArnT, allowing me to determine the chemical and structural mechanisms behind each step of their respective catalytic cycles through combined classical and quantum mechanics. My current focus is on the use of emerging techniques to study how the dynamics of these enzymes change over time in response to perturbations such as pH or substrate binding, allowing me to determine functionally important residues and track the propagation of signals throughout the protein to identify allosteric, therefore druggable, binding sites. Essentially, this means that I can identify specific areas which could bind small molecular inhibitors to prevent functionally important dynamic changes, allowing me to potentially design lead molecules for codrugs which could reverse polymyxin resistance. Excitingly, this project will allow me to work in vitro so that I can iteratively use my computational work to inform and design tests on these enzymes in real life, studying specific mutants and determining the chemical groups and conditions responsible for certain mechanisms for each step of the cycle, which I can then use to improve my work in silico. Multidrug resistance is currently estimated to kill nearly 5 million people per year; my major aim for this work is to allow significant progress in our understanding of how we can treat the most severe cases of resistant bacterial infections and to hopefully go some way to help develop medicine which could be used to overcome this emerging crisis. Antibiotic resistant bacteria inside a biofilm | iStock.com/Dr_Microbe 25
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Eagle Eye Magazine Issue 1 2023

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