The research described in this newsletter is supported as part of  Biological Electron Transfer and Catalysis, an Energy Frontier Research Center funded by the U.S. Department of Energy, Office of Science.
BETCy researchers publish in Nature, Biochemistry, BBA and JBC

Director John Peters named fellow of AAM and AAAS
Biological methane production from bacterial iron-only nitrogenase  

Methane (CH 4 ) is a potent greenhouse gas, roughly 30 times more potent than carbon dioxide (CO 2 ). CH 4 is released from fossil fuels and is also produced by microbial activity, with at least one billion tons of CH 4 being formed and consumed by microorganisms in a single year. Complex methanogenesis pathways used by archaea have long been believed to be the exclusive route for CH4 production in nature.
A recent
paper by BETCy researchers describing a new pathway for methane production using bacterial Fe-only nitrogenase was published in the Jan. 15, 2018 issue of the journal Nature Microbiology. The work is also highlighted in a News and Views article in the same issue of the journal.
This paper reveals that Fe-only nitrogenase converts nitrogen gas to ammonia and CO2 into CH4 at the same time. This discovery is significant because it represents a potential new source of methane, a major part of the carbon cycle and a major greenhouse gas, in the environment. Fe-only nitrogenase represents one of the three different types of nitrogenases, which are each differentiated by the metal composition in the active site cofactor: MoFe, VFe, or FeFe. Though the flux of electrons for CO2 reduction to CH4 by Fe-only nitrogenase is relatively small, the amount of CH4 generated is enough to allow a methane-utilizing bacterium to grow in co-culture with a bacterium named Rhodopseudomonas palustris that is expressing Fe-only nitrogenase.
"The Fe-only nitrogenase has been a neglected enzyme, which is active in microbes more often and in more conditions than we had previously thought," said Yanning Zheng, lead author of the study, who is a senior fellow in BETCy's Harwood Laboratory at the University of Washington.
BETCy scientists with different backgrounds worked together on this project to examine the distribution of Fe-only nitrogenases in microbes and their functions both in vivo and in vitro. The interdisciplinary project was made possible by integrating microbiologists at BETCy labs at University of Washington, geobiologists at Montana State University, and enzymologists at Utah State University. This discovery gives us a new understanding of biological CH4 production in nature, the global carbon cycle, and the interactions in microbial communities.
Fe-nitrogenase active site FeFe-cofactor detailed in Biochemistry

In a second paper describing nitrogenase, BETCy research has illuminated key mechanistic details of the N2 reduction mechanism for the Fe-nitrogenase active site FeFe-cofactor. The work was published in the
Feb. 6, 2018 issue of the journal Biochemistry.
There are three forms of nitrogenase: Mo-nitrogenase, V-nitrogenase, and Fe-nitrogenase. While structurally similar, the forms differ in the metal content of their active site cofactors and are reported to reduce N2 at different rates with varying efficiency.
Fe-nitrogenase is the rarest and least understood of the forms, as well as being the poorest at N2 reduction. In the conventional Mo-nitrogenase, binding of N2 to the cofactor is known to follow a reductive elimination/oxidative addition (re/oa) mechanism. In this re/oa, two bridging Fe-H-Fe hydrides are reductively eliminated as N2 undergoes oxidative addition to the cofactor. A key feature of the mechanism is that it is reversible with hydrogen gas (H2), such that H2 effectively inhibits N2 reduction. Due to its differences from the Mo-nitrogenase, it was unclear if the Fe-nitrogenase would follow this same mechanism.

In this publication, it is demonstrated that Fe-nitrogenase also follows the re/oa mechanism for binding N2 to its cofactor. It is also shown that the enzyme has a relatively low affinity for N2 as a substrate, providing insight into its poor reduction activity.
Lead author Derek Harris, a graduate student in the Seefeldt Group at Utah State University, purified the component proteins of the Fe-nitrogenase from Azotobacter vinelandii. The system was biochemically characterized and tested for the ability of hydrogen gas (H2) to inhibit N2 reduction. H2 did inhibit N2 reduction and when presented with deuterium gas (D2) in place of H2, HD was detected by mass-spectrometry, confirming a reversible re/oa mechanism. N2 is reductively eliminated from the cofactor and D2 undergoes oxidative addition as two Fe-D-Fe deuterides. As the cofactor relaxes, the deuterides are released and coordinate with a proton to form HD.
In examining the partial pressure dependence of N2 on NH3 formation, Fe-nitrogenase displayed the same previously reported inefficiency (producing excess H2 per N2 reduced). However, it was also revealed that Fe-nitrogenase has a five-fold lower affinity for N2 than the Mo-nitrogenase. Because of this, it is difficult to put Fe-nitrogenase under a saturating concentration of N2. Extrapolating to what would be a saturating concentration Fe-nitrogenase should achieve the same efficiency as Mo-nitrogenase. This lowered affinity and efficiency is likely a result of the effect differing metals in the cofactor has on its electronics.
Overall, this work provides evidence for a unifying mechanism for N2 binding amongst the forms of nitrogenase. It also provides insights into how nature has evolved redundant systems to ensure that this crucial reaction is carried out, albeit with compromises.
John Peters earns dual fellowships with AAM and AAAS

BETCy director John Peters has been named a Fellow in the American Academy of Microbiology. This is Peters' second such honor in the last three months: last fall he was named a Fellow in the American Association for the Advancement of Science for his contributions in chemistry.

Peters also directs the
Institute of Biological Chemistry at Washington State University.  The American Academy of Microbiology represents the American Society for Microbiology , the world's oldest and largest life science organization. The mission of the Academy is to recognize scientists for outstanding contributions to microbiology and provide microbiological expertise in the service of science and the public.

"I hope the work I've done with my colleagues brings a better understanding of how living organisms use energy, leading to reduced fertilizer use or increased efficiency in our use of energy," Peters said. "This research we've done lays the groundwork to help grow crops or make energy production and utilization more efficient."

Peters said he also is proud of the recognition in two completely different fields: chemistry and microbiology. "It says a lot about the scope of our program and what we do to advance science," he said.

Peters' election as an  American Association for the Advancement of Science (AAAS) Fellow  is a distinction bestowed upon AAAS members by their peers, in recognition of scientifically or socially distinguished efforts to advance science and its applications.
"We've done some groundbreaking work that's laid the foundation for others to follow," Peters said. "That's the most rewarding part of my research, that a lot of significant science has been born out of the work my colleagues and I have done."
That foundation includes discovering how to make energy much more efficiently. For example, it has the potential, though it's still very early, to extract more energy from biomass when making biofuels, Peters said.
"It's an honor to be nominated by people that I respect so much," Peters said. "I'm greatly appreciative that they took the time and effort to nominate me and feel my work is valuable to our field."
BETCy researchers determine role of allostery in the model electron bifurcating enzyme, Nfn
Bifurcating enzymes control the direction of electron transfer through a process known as electron gating. In a recent publication, the BETCy team provides insight into the electron gating mechanism of the bifurcating Nfn enzyme through a combination of computational and biophysical methods.

The article "H/D exchange mass spectrometry and statistical coupling analysis reveal a role for allostery in a ferredoxin-dependent bifurcating transhydrogenase catalytic cycle
appeared in the January 2018 issue of Biochemica et Biophysica Acta. 

Luke Berry, a graduate student in the Bothner Lab at Montana State University, conducted hydrogen deuterium exchange coupled to mass spectrometry (HDX-MS) to investigate the conformational dynamics of Nfn as the enzyme proceeds through the catalytic cycle of electron bifurcation. Individual steps of the cycle were simulated by adding pyridine nucleotides and ferredoxin to Nfn in several different combinations. Upon substrate binding, the team observed that conformational changes occurred in distal regions of the Nfn enzyme complex, suggesting the presence of pathways for communication and a mechanism for electron gating.

This work was enabled by the wide range of expertise on the BETCy team. The Nfn protein complex was purified by  Gerrit Schut, a research scientist in the  Adams Lab at the  University of Georgia. Using a sophisticated statistical analysis of amino acid sequences,  Saroj Poudel, a graduate student in the  Boyd  Group at  Montana State University, identified functional residues conserved in over 400 Nfn-like proteins. These residues form physical networks, or pathways of communication through the protein.

This work shows that electron transfer down the endergonic and exergonic pathways in Nfn are regulated through the binding of different substrates, and identifies potential networks of allosteric communication within the enzyme complex. The connectivity illustrated by these networks allows Nfn to integrate input from multiple substrate and product concentrations and will likely play a critical role in how the enzyme controls the flow of electrons.
Probing the flavins in FixAB
One of the electron bifurcating enzymes examined by BETCy is the Fix system, which furnishes low potential electrons to nitrogenase. At the core of this system is the heterodimeric FixAB complex, which houses two FAD cofactors. One of the FADs is the presumed site of bifurcation while the other FAD is thought to act as an electron conduit. A key challenge has been the overlapping spectral signals of the two FADs.
In a recent paper, BETCy researchers at the University of Kentucky, the National Renewable Energy Lab and Montana State University have succeeded in isolating the FixAB complex and demonstrated a strategy for distinguishing the two FADs from one-another spectroscopically. This work was published in the March 30, 2018 issue of Journal of Biological Chemistry.

H. Diessel Duan (University of Kentucky) tested FixAB's adherence to the accepted model for bifurcation by characterizing the natures and energetics of each of the electron transfer reactions, and Cara Lubner (NREL) evaluated FixAB's capacity to access a transient semiquinone state that is thermodynamic suppressed.  BETCy's mass spectrometry expert (Monika Tokmina-Lukaszewska, Montana State University) confirmed the FixAB's integrity.

For the more reducing flavin, the semiquinone states are unstable, as required for optimal bifurcation activity, yet the potential still allows acceptance of a pair of electrons from NADH. However the higher-potential (electron transfer) flavin acts as a carrier of single electrons and the value of its higher potential indicates that it will be partially reduced at rest. This places a 'choke' on electron transfer along the exergonic path allowing only one electron to pass to the electron transfer flavin and thereby forcing the other one to follow the endergonic energy conserving path to ferredoxin.
On both thermodynamic and kinetic grounds, FixAB conforms to the models BETCy has refined in a variety of complementary bifurcating systems. However the relative simplicity of the FixAB system in conjunction with the authors identification of several new spectral signatures, opens the door to detailed mechanistic studies and examination of the roles of individual amino acids in supporting bifurcation, based on site-directed mutagenesis in this system that can now be produced in  E. coli.
Know someone who would like to get these announcements? Please forward this on! New subscribers can sign up online.
Visit us online at
or on Facebook