In this issue...
  • BETCy scientists describe light-driven conversion of greenhouse gas to fuel
  • BETCy scientists uncover rate-limiting step in nitrogenase catalytic cycle
  • BETCy publishes classification scheme for [FeFe]-hydrogenases
  • BETCy hosts second scientific advisory board meeting in Seattle Sept 28-29
  • BETCy scientists present at seminars and conferences
The research described in this newsletter is supported as part of the 
Biological Electron Transfer and Catalysis, an Energy Frontier Research Center funded by the 
U.S. Department of Energy, Office of Science.
Scientists from BETCy EFRC describe light-driven conversion of greenhouse gas to fuel
Discovery featured in Scientific American; paper citations are in top 5%

BETCy EFRC researchers have used a phototropic bacterium as a biocatalyst to generate methane from carbon dioxide in one enzymatic step. The team says the break-through puts them one step closer to cleanly converting harmful carbon dioxide emissions from fossil fuel combustion into usable fuels.

From left: Utah State University biochemists Sudipta Shaw, Derek Harris and Lance Seefeldt are part of the seven-institution US Department of Energy Office of Science's Energy Frontier Research Center program-funded Center for Biological and Electron Transfer and Catalysis (BETCy) collaboration. The multi-institution team generated methane from carbon dioxide in one enzymatic step. Credit: Mary-Ann Muffoletto, Utah State University
The lead scientists are located at Utah State University and the University of Washington. 
Using an engineered strain of the phototropic bacterium Rhodopseudomonas palustris as a biocatalyst, the researchers generated methane from carbon dioxide in one enzymatic step. A key feature was that cells expressed large amounts of a remodeled form of the enzyme nitrogenase and the catalysis was driven by light.

Their findings were published in the Proceedings of the National Academy of Sciences. Co-authors are BETCy principal investigators Lance Seefeldt (Utah State University) and Caroline Harwood (University of Washington) along with members of their research team Kathryn Fixen and Yanning Zheng of UW; Derek Harris, Sudipta Shaw and Zhi-Yong Yang of USU; and Dennis Dean of Virginia Tech.

"It's a baby step, but it's also a big step," said Seefeldt, a professor of chemistry and biochemistry at USU. "Imagine the far-reaching benefits of large-scale capture of environmentally damaging byproducts from burning fossils fuels and converting them to alternative fuels using light, which is abundant and clean."

In an article about the discovery published Aug. 23 in Scientific American, Harwood, a professor of microbiology, said the report blossomed from her work studying an enzyme called nitrogenase.
"We're really interested in the enzyme nitrogenase because it does a phenomenally difficult reaction," she said.
"Use of phototrophs opens a new world of possibilities," said Seefeldt, "These kinds of bacteria could be used to make not only fuel, but all kinds of materials we use in everyday life, without the use of environmentally harmful energy sources. The future of this research is incredible."

The original paper was published in the Proceedings of the National Academies of Science of the USA (PNAS): Light-driven carbon dioxide reduction to methane by nitrogenase in a photosynthetic bacterium doi: 10.1073/pnas.1611043113 
Attention given the PNAS paper places the article the top 5% of all research outputs scored by Altmetric, putting it in the 98th percentile of all articles its age. See data at
B ETCy scientists uncover rate-limiting step in nitrogenase catalytic cycle

In a recent publication, BETCy scientists demonstrated that the rate limiting step in the nitrogenase catalytic cycle is the release of inorganic phosphate. In the BETCy study, the physiological reductant flavodoxin was used to uncover the rate limiting step. This is in contrast to most earlier studies which used dithionite, a nonphysiological reductant, largely because of ease of use. The new study by BETCy puts a fresh perspective on the mechanism of biological nitrogen fixation as it occurs in vivo.

Nitrogen is a critical nutrient for all living organisms. Without it, plants wouldn't grow, humans would have no food and life would not exist. Although the majority of the Earth's atmosphere is made of up nitrogen gas (N 2), it cannot be used by most living organisms because of its high stability. Some microbes, however, produce the nitrogenase enzyme, which converts N 2 into ammonia (NH 3), the form of nitrogen that living organisms can utilize in their growth and metabolism.
The conversion of N 2 to NH 3 by nitrogenase in vivo is comprised of a series of steps using the energy of ATP hydrolysis to move reducing units from a reductant, flavodoxin or ferredoxin, to the catalytic center of the nitrogenase enzyme. To uncover the rate-limiting step of the reaction as it occurs in vivo, BETCy scientists measured the speed of each step in the reaction with the physiological reductant flavodoxin and compared it to the rate constants using dithionite. These experiments showed that Pi release is the slowest step of the nitrogenase catalytic cycle, a finding which is consistent with other ATP-hydrolyzing enzymes. The slowest step is important, because it limits how fast the overall reaction can go or, in other words, how fast NH 3 can be produced.
While scientists are still uncovering the intricacies of nitrogenase, this new work provides insight into one of the most difficult, yet most important reactions on the planet. This work was published in the article entitled, "Evidence that the Pi Release Event is the Rate Limiting Step in the Nitrogenase Catalytic Cycle, " in Biochemistry 55:3625-3635.

BETCy publishes classification scheme for [FeFe]-hydrogenases
A team of BETCy researchers led by PI Eric Boyd (Montana State University) recently published a new classification scheme for [FeFe]-hydrogenases. These hydrogenases (Hyd) are a structurally diverse class of enzymes that catalyze the reversible oxidation of hydrogen (H2).  Recently, several Hyd complexes have been shown to catalyze electron bifurcation (EB), whereby two electrons derived from the oxidation of (H2) are split, with one used in the exergonic reduction of the cofactor NAD+ and the other used to catalyze the endergonic reduction of ferredoxin. In the present study, they used bioinformatics approaches informed by biochemical data to identify the structural determinants of Hyd that underpin differences in enzyme functionality, with a particular as they relate to EB capability. 

Hyd homologs were clustered into one of the three classification groups, Group 1 (G1), Group 2 (G2), and Group 3 (G3). G1 enzymes were predicted to be monomeric while G2 and G3 were predicted to be multimeric and include HydB, HydC (G2/G3) and HydD (G3). Variation in the catalytic subunit of Hyd (HydA) did not change with the above group designations. Since all known Hyd with bifurcation capability are G2/G3 enzymes, these results suggest that bifurcation capability is dictated primarily by the presence of both HydB and HydC in Hyd complexes. The classification scheme and neighboring protein encoding genes identified herein provides a framework for future biochemical and mutagenesis studies to elucidate the functional role of Hyd enzymes.
This work was published in the article entitled, "Unification of [FeFe]-hydrogenases into three structural and functional groups," in Biochim. Biophys. Acta, Gen. Subj., 1860:1910-1921.

BETCy hosts second scientific advisory board meeting

BETCy hosted its second meeting  with its Scientific Advisory Board in Seattle on Sept. 28-29, 2016. The meeting's agenda included a recap of accomplishments and publications, and it provided a forum to lay plans and set priorities for the next year and beyond.

Director John Peters opened the meeting by thanking all PIs and key personnel for attending and then set forth a busy schedule for the two day meeting. On the morning of Wednesday, Sept. 28, the lead PIs from each of the Center's three thrusts gave an overview of the highlights from the past year. The PIs and key personnel then met to go over plans for the coming year. Later in the afternoon, the thrust leaders presented their outlook for future research to the entire team.

On Wednesday evening, the advisory board joined the team for dinner and a poster session led by the key personnel. On Thursday morning, Sept. 29, the thrust leaders presented an executive summary to the board, which included both new research developments from the past year and the plans for the coming year. Later in the morning, the advisory board met privately for discussion and to prepare their feedback to the BETCy team.

At the conclusion of the meeting, Dr. Greg Ferry, chair of the advisory board, told the group that it is clear that BETCy has accomplished much since the 2015 meeting in Athens, Georgia, and that the board is impressed with the ambitious research plans presented by thrust leaders.

Advisory board members in attendance at the Seattle meeting included Robert Blankenship (Washington University), Greg Ferry (Penn State University), Mary Lidstrom (University of Washington), Bill Metcalf (University of Illinois), and Mike Seibert (National Renewable Energy Laboratory). In addition, BETCy hosted 2 additional board members for the meeting: Russ Hille (University of California at Riverside) and John Leigh (University of Washington).

BETCy researchers present at seminars and conferences
Since the last issue of this newsletter, BETCy research has been presented at the following conferences:

Caroline Harwood presented a seminar entitled, "Tackling thermodynamics: coaxing bacteria to release energy-rich compounds."  Seminar was given at the Dept. of Molecular Genetics and Microbiology, Michigan State University. Sept. 13, 2016.
Michael Adams presented a plenary lecture entitled "Structure, Function and Evolution of Hydrogen-Evolving, Energy-Conserving Hydrogenases from Hyperthermophiles" at the Extremophiles Conference, Kyoto, Japan, Sept. 12-16, 2016.
Paul King presented a seminar at the Department of Chemistry, University of California-Davis, Sept. 4, 2016.

Paul King presented the keynote talk at the 12th Forum Chemical Engineering in Coatzacoalcos, Mexico. September 2016.

Two members of the Seefelt group presented posters at the Hansen Life Sciences Retreat, Utah State University, Logan, UT, September 2016. Derek Harris presented "Fe-only Nitrogenase," and Rhesa Ledbetter presented "Purification and Characterization of the Electron Bifurcating FixABCX Complex from Azotobacter vinelandii.

David Jennings from the Jones group presented a poster entitled "Electrocatalytic Activity of the Electron Bifurcating Enzyme NADH-dependent Reduced Ferredoxin:NADP+ Oxidoreductase (Nfn)" at the 13th European Biological Inorganic Chemistry Conference, Budapest, Hungary, August 2016.
Cara Lubner from the King group was selected from a poster competition to present the talk "Resolving the Mechanism of Flavin-Based Electron Bifurcation" at the Gordon Research Conference on Electron Donor Acceptor Interactions in Newport, RI, August 7-12, 2016.

David Mulder from the King group presented a talk and poster, "Mechanistic Investigations on Electron Bifurcation by EPR Spectroscopy" at the EPR Symposium as part of the Rocky Mountain Conference on Magnetic Resonance, Breckenridge, CO, July 17-21, 2016.

Several BETCy scientists presented at the 11th International Hydrogenase Conference, Marseille, France, July 10-14, 2016. Anne Jones presented a seminar entitled, "Defining Functional Diversity of [FeFe]-Hydrogenases Electrochemically." Jacob Artz from the Peters group presented a poster entitled, "Structural Determinants of Catalytic Bias in [FeFe]-Hydrogenases as Revealed by Potentiometric EPR Spectral Deconvolution," and Saroj Poudel from the Boyd group presented a poster entitled, "Unification of [FeFe]-Hydrogenases into Three Structural and Functional Groups."

John Hoben from the Miller group presented a poster entitled "Fleeting Flavin Intermediates: Understanding Lifetimes Ranging over Three Orders of Magnitude in Terms of Electron Transfer and Charge Recombination" at the Gordon Research Conference on Enzymes, Coenzymes and Metabolic Pathways in Waterville Valley NH, 24-29 July 2016 and the associated Gordon Research Seminar on July 23-24. 
Several members of the Peters group presented at the Northwest Crystallography Workshop, in Pullman, WA, June 17-19, 2016: Jacob Artz presented a poster entitled, "Structural determinants of catalytic bias in [FeFe]-hydrogenases as revealed by potentiometric EPR spectral deconvolution," Jacquelyn Miller presented a poster entitled, "Structural determinants of bifurcation in energy conserving electron transferring flavoproteins," Natasha Pence presented a poster entitled, "Identifying protein-protein interactions that facilitate electron transfer between the nitrogenase Fe protein and its physiological reductant flavodoxin from Azotobacter vinelandii," and Oleg Zadvornyy gave a seminar entitled, "Electron bifucation: structural insights into NADH-dependent ferredoxin NADP oxidoreductase (NfnI) from Pyrococcus furiosis."

Paul King presented a poster at the poster at the GRC on Metallocofactors in Easton, MA, June 12-17, 2016.
Monika Tokmina-Lukaszewska from the Bothner group presented the talk "Characterization of a Nitogenase Complex from Azotobacter Vinelandii in Gas Phase Using Native Mass Spectrometry and Ion Mobility" at the 64th Conference on Mass Spectrometry and Allied Topics in San Antonio, TX, Jun 5-9 2016.
Kathryn Fixen from the Harwood group presented a seminar entitled "Shining Light on How a Photosynthetic Bacterium Works" at the University of Washington, June 2, 2016, Seattle, WA.
Garrett Williams from the Jones group presented "Determinants of Catalytic Bias in [FeFe]-Hydrogenases" at the Penn State Bioinorganic Workshop, June 2 - 10, 2016.
Two members of the Adams group presented at the University of Georgia Dept. of Biochemistry and Molecular Biology Retreat at Unicoi State Park, GA, May 15-16, 2016. Gerrit Schut presented a poster entitled, "Bifurcating Hydrogenases - the Role of Electron Transfer Conduits in Gating Electron Transfer," and Diep Nguyen presented a poster entitled, "Bifurcating Nfn - a Simplified Model System to Examine the Structural Basis for Electron Bifurcation."