Previously, it was not possible to capture high-resolution images of nitrogenase, the only enzyme capable of reducing nitrogen to ammonia during catalysis. Now, researchers at the University of California, San Diego, report the first near-atomic resolution snapshot of nitrogenase during catalysis using cryoelectron microscopy (cryoEM).The result was published in a magazine chemistry.
This research was accomplished through a close partnership between the groups of Professor Akif Tezcan and Assistant Professor Mark Herzik of the Department of Chemistry and Biochemistry at the University of California, San Diego. Tezcan has been researching for a long time, nitrogenaseHerzik provided the cryo-EM expertise necessary to perform the study.
“This is a very important biological advance. nitrogen Tezcan said: “It is very interesting to be able to obtain atomic resolution images of enzymes as dynamic and complex as nitrogenase in action. paves the way for a full understanding of
To understand the importance of these cryo-EM images, we need to understand the tremendous global importance of nitrogen fixation. All organisms require a ‘fixed’ nitrogen source for the biosynthesis of the building blocks of life such as proteins and DNA. However, most organisms lack the nitrogenase enzyme and are unable to metabolize atmospheric nitrogen into bioprocessable forms.
Nitrogenase was essentially the only source of fixed nitrogen in the biosphere until the advent of the Haber-Bosch process, an industrial method for converting atmospheric nitrogen to ammonia, more than 100 years ago. Industrially produced ammonia was primarily used as a fertilizer, and its advent revolutionized his agricultural practices in the first half of the 20th century. The Haber-Bosch Act has often been cited as the driving force behind the world population explosion of the last 100 years that “turned air into bread.”
However, the Haber-Bosch process is very energy intensive, requiring temperatures in excess of 400°C and high pressures of hydrogen gas. An estimated 1-2% of global energy production is consumed by the Haber-Bosch process. This process also raises environmental concerns, such as nitrate leaching into groundwater and increased greenhouse gas emissions. nitrous oxide.
A key issue driving biological nitrogen fixation research is the contrast between nitrogenase and the Haber-Bosch process. Industrial processes require such extreme conditions, so how do enzymes catalyze nitrogen reduction at ambient temperature and pressure?
“If we can understand the mechanism of nitrogenase, we can not only understand why nature evolved nitrogenase into such a complex enzyme, but also design principles for producing ammonia in a more cost-effective and environmentally friendly way. It may become clear,” he said. Tezukan.
Much is known about the structure of nitrogenase, but largely due to technical limitations, until now, atomic resolution imaging of the enzyme during its “inversion” or in the process of catalyzing atmospheric nitrogen to ammonia has been impossible. No one was able to obtain it.
Scientists can use X-ray crystallography to obtain atomic resolution images of proteins, but this method requires the protein to be fixed in place within the crystal, in a sense stationary. there is. In other words, nitrogenase in action cannot be captured. Nitrogenase catalysis requires different parts of the enzyme to bind together and break down several times to make a single enzyme. ammonia Molecule from Nitrogen. The process is not stationary.
CryoEM not only allows researchers to capture structures without the need to immobilize proteins on crystals, but thanks to recent advances in hardware and data processing, it can do so at atomic resolution. Such high resolution is necessary to visualize small changes associated with enzyme catalysis.
These advances prompted Tezcan and graduate student Hannah Rutledge to consider using cryoEM to study nitrogenase in catalysis. For this, they enlisted the help of resident cryo-EM expert Mark Herzik and his group members Brian Cook and Hoang Nguyen.
“This was an exciting and technically challenging project to pursue during the pandemic. While cryoEM is a very capable technology, very little research has been reported on when enzymes are catalyzed. “An important insight and technology development in this study is that it paves the way for future investigations not only of nitrogenase mechanisms, but of enzymes in general,” said Herzik.
Herzik and Rutledge have worked closely together to prepare hundreds of CryoEM samples. Since nitrogenase is oxygen sensitive, samples were prepared in an anaerobic glovebox, quickly transferred and frozen within seconds to prevent degradation. Ultimately, the team collected over 15,000 videos of him capturing over 20 million individual molecules at various stages of catalysis.
It took the team almost a year to organize the terabytes of data. Low quality images were discarded and all particles were identified and classified. Finally, they were able to obtain the first atomic resolution images of nitrogenase in the middle of its turnover.
The cryo-EM structure revealed several unexpected features of nitrogenase not previously observed in the X-ray structure. Importantly, the new observations provide new mechanistic hypotheses for nitrogenase catalysis. Tezcan and Herzik hope to collaborate for many years to test these hypotheses and to understand in detail the catalytic mechanisms of nitrogenases.
“This is just the beginning,” said Tezcan. “I can see the big picture enzyme Now, not just one particular part, but upon catalysis. This really opens the floodgates for further research to understand how nitrogenase works and, in the future, may develop more efficient processes to generate fixed nitrogen. ”
Hannah L. Rutledge et al, Structure of a nitrogenase complex prepared under catalytic rotation conditions, chemistry (2022). DOI: 10.1126/science.abq7641. www.science.org/doi/10.1126/science.abq7641
University of California, San Diego
Quote: Researchers Acquire First Ever Cryo-EM Image of Nitrogenase in Action (July 28, 2022) https://phys.org/news/2022-07-first-ever-cryo-em- Retrieved 07/28/2022 from images-nitrogenase-action.html
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Researchers take first-ever cryo-EM images of nitrogenase in action
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