Atomic-resolution images could open a new path to ammonia production

Previously. the only enzyme capable of reducing nitrogen to ammonia, during the catalytic action. Now, for the first time, researchers at the University of California, San Diego are reporting near-atomic resolution snapshots of nitrogenase during catalysis using cryogenic electron microscopy (cryoEM). The results have been published in the journal Science.

This work was accomplished through a close partnership between the groups of Professor Akif Tezcan and Professor assistant Mark Herzik, both from the Department of Chemistry and Biochemistry at UC San Diego. While Tezcan has long studied nitrogenase, Herzik provided the cryoEM expertise needed to carry out the research.

“This is a very important step forward in terms of biological nitrogen fixation as well as structural biology, in general,” Tezcan said. This opens the door to a full understanding of the mechanism of this enigmatic enzyme, which has preoccupied researchers for decades.”

All organisms need “fixed” sources of nitrogen for the biosynthesis of the building blocks of life such as proteins and DNA. However, most living organisms lack the enzyme nitrogenase and cannot metabolize atmospheric nitrogen into a biotransformable form.

Nitrogenase was essentially the only source of fixed nitrogen in the biosphere until the advent of the Haber-Bosch process — the industrial procedure for converting atmospheric nitrogen into ammonia — over a hundred years ago. Industrially produced ammonia is widely used for fertilizers and its advent revolutionized agricultural practices in the first half of the 20th century. The Haber-Bosch process has often been cited as the driving force behind the global population explosion over the past century, having “turned air into bread”.

However, the Haber-Bosch process is very energy-intensive, requiring temperatures above 0093°C and high pressures of hydrogen gas. It is estimated that 1-2% of all the world’s energy output is consumed by the Haber-Bosch process. The process also raises environmental concerns, including the leaching of nitrates into groundwater and higher emissions of nitrous oxide, a greenhouse gas.

A key question that drives biological research on nitrogen fixation is the contrast between nitrogenase and the Haber-Bosch process. How does the enzyme catalyze the reduction of nitrogen at ambient temperature and pressure when the industrial process requires such extreme conditions?

“If we can understand the mechanism of nitrogenase, not only can we understand why nature has turned it into such a complex enzyme, but we might also discover principles of design for the production of ammonia in a more cost-effective and environmentally friendly way,” Tezcan said.

Although much is known about the structure of nitrogenase, so far. largely due to technological constraints.

This method requires proteins to be spot-fixed in a crystal – stationary in one sense – which means it cannot capture nitrogenase in action. Nitrogenase catalysis requires different parts of the enzyme to associate with each other and then separate several times to form a single molecule of ammonia from nitrogen. The process is anything but stationary.

CryoEM not only allows researchers to capture protein structures without them being fixed in crystals, but, thanks to recent advances in hardware and data processing, to do it with atomic resolution. Such high resolution is needed to visualize the small changes associated with enzyme catalysis.

These advances led Tezcan and graduate student Hannah Rutledge to consider using cryoEM to study nitrogenase in catalytic motion. And for that, they enlisted the help of resident cryoEM expert Mark Herzik and his group members Brian Cook dinner and Hoang Nguyen.

“It was both an exciting project and a technological challenge to pursue, during the pandemic no less. Although cryoEM is a very powerful method, few studies have reported on enzymes as they undergo catalysis. The critical insights and technological developments from this study not only pave the way for future explorations of the mechanism of nitrogenase but of enzymes in general,” said Herzik.

Herzik and Rutledge worked closely to prepare hundreds of cryoEM samples. Because nitrogenase is sensitive to oxygen, samples were prepared in an anaerobic glove box, then quickly transferred and frozen within seconds to prevent degradation. In the ultimate, the team collected more than 15 000 videos capturing over 18 millions of individual molecules at various stages of catalysis.

It took the teams almost a year to sort through several terabytes of data: they discarded the poor quality images, then identified and classified all the particles. Finally.

The cryoEM buildings revealed several unexpected features of nitrogenase that were previously not observed in X-ray buildings. Significantly, the new observations provide a new hypothesis mechanistic for nitrogenase catalysis. Tezcan and Herzik hope to collaborate for many more years to test these hypotheses and understand in detail the catalytic mechanism of nitrogenase.

“This is just the beginning,” said Tezcan . “We now have a picture of the entire enzyme, not just a specific part, during catalytic action. This will really open the floodgates for further research to understand how nitrogenase works and potentially later develop more efficient processes to produce fixed compounds. nitrogen.”

Funding provided by the National Institutes of Health grants R-GM099813, R35-GM099813 and T32-GM008326 NASA grant 80NSSC18M0093 and the Searle Fellowship Program. CryoEM experiments were conducted at the CryoEM facilities at UC San Diego as well as the Stanford-SLAC Cryo-EM Heart.

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