Why some nitrogen-processing enzymes are more efficient than others
Nitrogen gas is abundant in Earth’s atmosphere, but most living organisms can’t readily use this nitrogen. Only a subset of microbes that have enzymes known as nitrogenases can break nitrogen gas apart and convert it into ammonia.
There are three different classes of nitrogenases found in nitrogen-fixing microbes, which vary based on the types of metal that they contain. Nitrogenases that contain the metal molybdenum are the most efficient, and two new studies from MIT offer an explanation for why that is.
The findings could help guide the design of engineered enzymes or synthetic catalysts that can convert nitrogen gas to ammonia, the researchers say.
The team found that while molybdenum doesn’t directly bind to nitrogen, it helps nearby iron atoms bind to nitrogen more strongly. This is a critical first step in breaking the bond between the two nitrogen atoms that form nitrogen gas.
“It’s that initial binding step that’s really the hard part. Once you’ve started to break the nitrogen-nitrogen triple bond and make some new nitrogen-hydrogen bonds, it’s pretty easy to get the rest of the way,” says Daniel Suess, the Arthur Amos Noyes Associate Professor of Chemistry at MIT and a senior author of both papers.
MIT postdoc Tong Wu and former postdoc Madeleine Ehweiner are the lead authors of one of the papers, and Alexandra Brown PhD ’23 is the lead author of the other. Kyle Lancaster, a professor of chemistry at Cornell University, is a senior author of the latter paper, along with Suess. Both papers appear today in the journal Chem.
Efficient enzymes
Before microbes evolved the ability to fix nitrogen around 3 billion years ago, the strong triple bond between atoms of N2 could only be split with high-energy events such as a lightning strike.
“Once an enzyme came along that could convert dinitrogen to ammonia, that changed the game because now cells could harvest nitrogen from the air for biomass,” Suess says.
Within the active site of nitrogenase is a catalytic cofactor that typically consists of a cluster of iron, sulfur, carbon, and in some cases another metal. Nitrogenases whose cofactors contain molybdenum are the most efficient, followed by those containing the metal vanadium. Nitrogenases that don’t have any metal other than iron are the least efficient.
Why the molybdenum-containing enzyme is more efficient has been a puzzle, especially because it’s thought that molybdenum itself doesn’t bind directly to nitrogen gas.
“In all cases, iron is thought to interact with N2, so it’s a bit of a mystery,” Suess says. “If all the chemistry is happening at iron, why is it that this molybdenum is affecting catalysis?”
To answer that question, Suess’s lab has developed simpler versions of iron-sulfur clusters that they can use to model the naturally occurring cofactors. These can be modified by adding different metal atoms, allowing the researchers to study how those metals change the cofactors’ properties.
In the first paper, led by Wu and Ehweiner, the researchers swapped in different metal atoms and then measured the ability of the iron in the cofactor to bind to nitrogen. They found that only cofactors with a large metal atom, such as molybdenum or tungsten, were able to strongly bind N2. With vanadium, chromium, or iron, which are smaller, the cofactors did not bind N2 and performed other reactions instead.
“That paper essentially recapitulates what you see in biology, which is that the iron-sulfur clusters that have molybdenum in them seem to be better at binding dinitrogen than those with lighter metals,” Suess says.
Sharing electrons
In the second paper, led by Brown, the researchers uncovered a possible mechanism that explains that phenomenon.
In that paper, the researchers studied how cofactors containing different metals interact with compounds called N-heterocyclic carbenes. These molecules behave similarly to N2 in some ways, making them a good model for this type of study. Like N2, they are resistant to accepting any electrons from another molecule, which is an essential step to breaking chemical bonds.
The researchers found that when molybdenum was included in the cluster, it became easier for iron to donate some of its electrons to the N-heterocyclic carbenes, in a process known as back-bonding. This occurs because molybdenum, a large atom, has large orbitals that can overlap with the orbitals of the nearby iron atom. That alters iron’s electron density in ways that make it easier for iron to pass electrons to N2.
“Without these direct metal-metal interactions, the iron has to do all the work, but adding the molybdenum allows for this electronic cooperativity,” Suess says.
Once N2 is bound to an iron atom, the rest of the reaction can proceed. A proton can come in from water or another source to create an N-H bond, which then makes it much easier for the remaining N-N bonds to be broken and bind to protons, forming NH3.
The findings could help guide scientists who are working on designing enzymes that could be engineered into organisms that help them generate their own NH3, eliminating or reducing the need for fertilizer. The results could also help chemists to design synthetic catalysts that could produce ammonia industrially, using less energy than the Haber-Bosch process.
“The general principle is that you can make an iron site in any context behave differently when you have these metal-metal interactions than when you don’t have these interactions,” Suess says. “The primary result of these findings is to teach us about the natural world and how nature accomplishes this really important and miraculous reaction. And, maybe that can be translated into new processes.”
The research was funded primarily by the U.S. Department of Energy, the National Science Foundation, and the National Institute of General Medical Sciences.