22. Jan. 2024

Brazilian researcher Dr. Vinicius Santana, a junior researcher from Magneto-Optical and THz Spectroscopy research group at CEITEC BUT, collaborated with biologists and chemists to explain a bioluminescence mechanism. The findings were published in a highly acclaimed study in the Nature Catalysis magazine. The groundbreaking technique Electron Paramagnetic Resonance (EPR) Spectroscopy has played a pivotal role in understanding catalytic reactions. This holds promise for green technologies. Group leader Assoc. Prof. Petr Neugebauer emphasizes their commitment to advancing and innovating EPR methodologies and techniques while actively participating in various applications and collaborations. He would also like to attract more students and workers to the field, where he sees huge potential. They could even join a new research group to be led by Vinicius Santana, who has been awarded the prestigious JUNIOR STAR 2024 grant to set it up.


How can we imagine the principle of EPR spectroscopy?

PN: The electrons play a key role. Atomic structures can be likened to a solar system where the nucleus is the sun, electrons are planets. Depending on what atom you have, you get a different planet constellation. Interactions between atoms, similar to different solar systems, involve electron behaviour, which plays a crucial role in chemical reactions and the formation of bonds. In EPR spectroscopy we use magnetic fields and microwave/radiofrequency radiation to excite these electrons and understand their surroundings. In many chemical reactions, electrons rearrange very fast, so you must play some tricks (like spin trapping EPR measurement) in order to capture it. We need to do that to understand these processes, especially in catalytic research where there is significant potential for future applications.

VS: This is technically called the charge transfer. You have the electron going from one species to another species and changing the chemical bonds within or between different reagents in this reaction. You don't have many techniques that can track these fast reactions and directly see them happening. With EPR we trap this electron in a species that is a bit more stable, we can then see the species, describe how it works, and then understand the real mechanism in this charge transfer. And by understanding the mechanism, you can then design new and better systems, which is what they did here.


Does it mean you want to promote the EPR technique in other catalytic research projects?

PN: Yes, we are now creating a network of experts in this field. The goal is to promote this technique in research, particularly in line with green technologies. Catalysis, which involves reducing energy to obtain a product, plays a crucial role. Compared to other methods requiring high temperatures and energy consumption, catalysis offers a more straightforward and energy-efficient way to reach the final product, ultimately reducing costs.


Let’s talk about the paper. I assume you were asked to do the measurement for your biology colleagues?

VS: When it is a big paper, it's usually put together by means of collaboration of many people. You need different kinds of expertise to solve this puzzle. Despite the fact that our colleagues had almost everything ready for the story but they were missing one key mechanism in one particular part of their reaction to understand how it works. So, they needed EPR to elucidate this particular part of the chemical reaction. We ran the measurements and helped them solve this problem, provide the last piece of the puzzle and say: okay, this is the reason why this particular pathway is going in this reaction and not some others.


They wanted to understand a specific catalytic mechanism – bioluminescence. Can you tell us more?

VS: Yes. In nature, enzymatic reactions play a crucial role. Enzymes, acting as catalysts, accelerate chemical reactions. Visualize it as having a specific key and lock pair. The substrate, akin to the key, is a unique molecule that fits into the enzyme lock, catalysing the reaction. Imagine it like a key fitting into a lock; only a particular molecule, the substrate, can bond with the enzyme, accelerating the reaction. Specifically, they used a substrate called coelenterazine which binds to luciferase, the enzyme responsible for speeding up the chemical reaction through charge transfer. When this reaction takes place, it results in an excited state that subsequently emits light at a specific wavelength. The colour of the emitted light is determined by the energy of this excited state.

They not only studied this enzyme but also engineered various versions derived from the Renilla coral species. They crafted an ancient version of the enzyme, characterized by stability and easy crystallization. This allowed them to elucidate the complete structure and observe how the substrate, acting as a key, seamlessly enters the enzyme, acting as a lock, thereby catalysing a rapid reaction. So, you drop the substrate into the solution with the enzyme, you immediately see it glowing very beautifully. This means that the reaction is happening, it's happening very fast and effectively. But this substrate itself also goes through this luminescence reaction. When it goes alone, it's called chemiluminescence. And when it goes into the enzyme, it's called bioluminescence.


So, you couldn’t track the reaction because of the speed?

Exactly. It's really hard to get inside and see what's going on. And as this charge transfer is very fast, there is no way we can apply a reporter, which is this molecule that will capture the electron and tell us with the EPR what's going on with the reaction.  So, it's not very easy to do that, maybe even impossible. We tried, but we didn't see any results. What we had to use was the chemiluminescence effect, which is the normal reaction without the acceleration from the enzyme. So, we watched this molecule when it was luminescent, but in the slow mode, let's say, because we didn't have the enzyme. And in this slower reaction, we could use this reporter to get this electron and to tell us how this chemical reaction is happening.


Can you describe your part of the work?

Our goal was to precisely determine if the reaction occurred via a radical route and, if so, identify chemical species formed in the reaction. We had to test different concentrations of each constituent and product in the reaction, aiming for the optimal yield of radicals (chemical species with unpaired electrons) that we could capture and subsequently observe with the EPR. To illustrate, the obtained spectra were not initially intense; the signal on the spectrometer was relatively weak. Consequently, we had to record and accumulate successive spectra (in this case 36 times), creating an average to extract the signal from the noise, allowing us to describe it effectively. Once we have the signal, the subsequent challenge involves analysis. We must fit and simulate this signal based on the potential chemical structure of the expected radicals. This process enables us to decipher the information embedded in the signal, such as details about the radical produced in that specific reaction.


How time-consuming was it?

Well… We tried different types of reporters and different types of reactions and concentrations until we got the ideal conditions to observe the chemiluminescence and get the EPR signal changes. This comprehensive analysis, including fitting and simulation, typically takes a bit longer than a week. After this, there is a quite extensive discussion phase. Despite having been measured when many aspects were already known, it still took approximately a year from the initial measurement to the eventual publication of the findings.


The paper was very well received. How do you feel about it?

It's really nice. It was a wonderful collaboration with these people. And I am very happy to be able to help people to answer their questions. It was simple, but they needed to answer what was going on in this particular part.


Can you explain the benefit of this study?

Scientists have been trying to explain the process of this particular bioluminescence reaction for the last 40 years, without success. The leading researchers in this study succeeded and we are happy to be part of the collaboration that unveiled it. And it opens the possibility of using the findings to replace conventional light bulbs. The new lighting options would be sustainable, energy-efficient and environmentally friendly. But there is still a long way to go.


How hard is it to communicate with experts in other fields and “build” a paper together? When you provide measurements to your collaborators, is the information clear to them, and can they effectively interpret the measurements?

VS: I have worked with physical chemistry since maybe my master’s. So, I understand a bit of chemistry. Then I can, not the specific parts of what they are telling me, but I can understand what they need. And then I can tell them: look, if you have this kind of reaction, this is how we can help. We can use this reporter, these traps, and make the measurements. And then I propose what to focus on, what trap to use, and then they proceed to do all the things I recommended. They cannot imagine what my simulations mean because EPR is a very specific technique. So, we provide the results individually, and then we have a discussion together.

PN: The measurement itself on the spectrometer doesn’t take long but to understand what's going on in the spectra, fitting it correctly, and making everything match can be a very time-consuming task. It can take to infinity. I have some data that nobody has understood up to now. It's just because it's too complex.


Do these unfinished things haunt you?

PN: No, no. This is something that you should kind of learn not to get bothered about. Electron Paramagnetic Resonance (EPR) reveals a captivating underside. It unveils the intricacies of nature, showcasing its beauty and the fascinating self-assembly processes. The complexity of these structures emphasizes how much remains unknown. The key reactions involving electrons, while crucial, highlight our current limitations in understanding. There's a concern that delving too deep into these processes without complete comprehension might pose risks, possibly leading to self-destruction.


Vinicius, what are you working on now?

I cooperate with many colleagues in Brazil, Argentina, Italy, Slovakia and, of course, within the Czech Republic. I mostly measure and analyse EPR data in different frequency ranges, to solve problems in several fields, from explaining reaction mechanisms, describing defects in oxides and identifying dopants to characterizing the intrinsic magnetic properties of molecular magnetic materials.

As for my main interest, I submitted a project to GACR Junior Star that was recently approved for funding, and I will start to lead a team in Jan/2024 to study newly synthesized molecular crystals. My team and I will investigate intra and intermolecular magnetic interactions and their correlations with the structure of these compounds, using the orientation of an external magnetic field to observe exotic phenomena such as quantum phase transitions and entanglement in these materials. The long-term goal of these studies is to produce and characterize new molecules that can have a potential application as quantum bits (qubits), which are the basic units of a quantum computer.