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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.
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 BETCy Web-based Tracking System in Beta-testing Phase
For several months the BETCy-EFRC has been developing a new web-based tracking system to catalog and report progress on specific protein targets that will help us unlock fundamental scientific questions about bifurcation, catalytic bias and electron flow. A beta version of BETCy Project Reporting & Information Database, or BETCy PRIDe, was launched center-wide for internal testing and training.
The web site is mobile friendly and uses a knowledge-centric workflow designed around the BETCy Google Drive data archive. Our new hybrid technology allows BETCy members to create unique protein tracking IDs center-wide and integrate them into various analytical data and/or knowledge streams on the cloud - providing team members with instant access to all BETCy knowledge about a given protein target (and scientific question).
This new model leverages the power of cloud storage but in a structured way that greatly improves the data sharing and knowledge mining within large-distributed research centers like BETCy. The initial production release for BETCy PRIDe is scheduled for October 2015, with new features to be added quarterly if needed.
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BETCy researchers present across the globe
Researchers affiliated with the BETCy EFRC have been giving presentations across the globe, from China to Chile and from Mississippi to Vermont. Below is a sampling of talks and presenters.
Michael Adams presented the invited talk entitled "Engineering a Hyperthermophilic Archaeon to Produce Fuels and Chemicals"on March 11, 2015 at NREL, Golden, CO.
Warintra Pitsawong and Anne-Frances Miller presented "Isotope Effects and Kinetic Mechanism of
Enterobacter cloacae Nitroreductase" at the American Society of Biochemistry and Molecular Biology, Experimental Biology meeting March 28 - April 1, 2015 in Boston MA.
Warintra Pitsawong and Anne-Frances Miller presented "H Transfer Coupled to e- Transfer in Nitroreductase" at the Southeast Enzyme Conference, April 11, 2015, Atlanta GA.
Michael Adams presented an invited talk entitled "Engineering Hyperthermophiles to Produce Fuels and Chemicals", Beijing University of Chemical Technology, Beijing, May 20, 2015.
http://www.buct.edu.cn/
Michael Adams presented an invited talk entitled "Engineering Hyperthermophiles to Produce Fuels and Chemicals", Shanghai Jiao Tong University, Shanghai, June 5, 2015.
John Peters presented the talk entitled "Evolution of modern respiration", at the Northwest Regional ACS Meeting in Pocatello, ID, June 21 - 24, 2015,
http://norm2015.sites.acs.org/
Eric Boyd presented "New Insights into the Evolution of Biological Nitrogen Fixation" at the GRC Cell Biology of Metals, July 2015, West Dover, Vermont.
https://www.grc.org/programs.aspx?id=13326
Anne-Frances Miller presented "One Man's Trash Is an Enzyme's Substrate: Mechanistic and Structural Features of a Promiscuous Enzyme" at the Gordon Research Conference on Enzymes, Coenzymes and Metabolic Pathways. July 12-17, 2015, Waterville Valley NH.
https://www.grc.org/programs.aspx?id=11264
Saroj Poudel
(Boyd Group) presented "Identifying the structural determinants that influence the directionality and function of [FeFe]-hydrogenase" at the GRC Cell Biology of Metals, July 2015, West Dover, Vermont.
https://www.grc.org/programs.aspx?id=13326
Michael Adams
presented an invited talk entitled "Engineering Thermophiles to Produce Liquid Fuels", Thermophiles 2015 Conference, Aug. 29, 2015, Santiago, Chile.
http://www.thermophiles2015.cl/welcome/
John Peters
presented at the Society for Industrial Microbiology Meeting in Philadelphia, PA, Aug. 4, 2015,
http://www.simbhq.org/annual/
John Peters
presented at the Thermophiles Conference in Santiago, Chile, Aug. 30 - Sept 4, 2015,
http://www.thermophiles2015.cl/welcome/
Anne-Frances Miller
presented "Flavins at the Foundation of Life" at the Midwest Enzyme Chemistry Conference. Sept. 12, 2015, Chicago IL.
http://www.midwestenzyme.org/
Anne-Frances Miller
presented "How proteins control electrons: protons" at the Department of Chemistry, Mississippi State University, Sept. 25, 2015.
http://www.chemistry.msstate.edu/
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AJ Rasmussen is first recipient of BETCy ETC Innovation award
AJ Rasmussen, who is a graduate student in the BETCy's Seefeldt research group, was the first recipient of the BETCy ETC Innovation award. Established by the Directors of BETCy, the purpose of ETC award is to
E
xploit
T
echnical
C
apabilities (ETC) and foster innovations and synergistic interactions between the 11 research groups in the BETCy EFRC. Applicants for the ETC award are asked to write a short proposal describing their proposed project, and awardees are granted supplementary travel funds to perform the research.
Mr. Rasmussen's work, described below, combines nitrogenase work in the Seefeldt laboratory at
Utah State University with electrochemistry techniques in the
Jones laboratory at
Arizona State University.
Developing a reductively stable electrode to allow direct electron transfer to nitrogenase
Nitrogenase, the enzyme responsible for reduction of the dinitrogen molecule to am monia, uses redox chemistry to perform nitrogen reduction catalysis. This means that electrons are transferred from an electron donor, the Fe protein component of nitrogenase in this case, to an electron acceptor, the MoFe component. These electrons are then used to reduce the dinitrogen bond, breaking it and forming ammonia. Understanding the redox chemistry of nitrogenase is key to elucidating the mechanism of nitrogen reduction. One way to better understand electron transfer in an enzyme is to link that enzyme to an electrode controlled by a potentiostat. Once this is accomplished it is possible to precisely control the flow of electrons into and out of the enzyme. The goal of this project is to create a link between the MoFe protein and an electrode so that we can further study the redox properties of nitrogenase.
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In September 2015,
John Hoben, a graduate student in the
Miller research group at the
University of Kentucky, received a BETCy ETC Innovation award. Mr. Hoben's work, described below, integrates the work of the Miller group with the
King research group at
NREL.
An Approach for Generating and Characterizing Highly Energetic Short-lived Intermediates
Enzymes that execute bifurcating electron transfer typically enlist a suite of protein bound small molecules to couple an energetically demanding electron transfer step to what is thought to be a rapid set of energy-releasing electron transfer steps. The series of electron transfer events likely involves short-lived intermediates, whose identification could provide much needed insight into the mechanism and control of electron transfer. To dat e, all bifurcating systems employed in low oxygen metabolism include at least one flavin protein site, although the systems vary in regards to the number of metal protein sites. However, it is not yet known how bifurcating enzymes tune flavin properties to promote this activity. The enzymes' fast turnover rates make mechanistic studies difficult.
The current September 2015 team building and capability expansion builds on data from a previously successful November 2014 visit, which established our ability to use transient absorption spectroscopy (TAS) to interpret flavin rapid decay kinetics.
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NREL installs advanced EPR instrument
Advanced Spin Resonance High Resolution Spectrometer.
The Advanced Spin Resonance Spectrometer (ASRS) located at NREL will bring cutting edge electron paramagnetic resonance (EPR) spectroscopic techniques to BETCy. The core of the ASRS is a Bruker ELEXSYS E580 with multi-frequency, multi-resonance, and variable temperature capabilities for probing electronic and molecular properties of Flavin and FeS electron transfer centers, and metalloenzyme catalytic sites. The multi-frequency capability (X- and Q-bands) along with pulse techniques will enable new approaches to systematically analyze and resolve complex EPR signals that arise from multiple paramagnetic centers found in bifurcating enzymes. Low-temperature (<80K) is required for EPR studies of metallo-clusters, and the ASRS is equipped with cryogen-free cryostats, providing on-demand access to temperatures of 4K.
The ASRS also will be outfitted with a variety of accessories, such as a goniometer for single crystal studies and a suite of resonators designed to maximize flexibility, sensitivity and experimental range. These include optical probes for light-triggered experiments that can be synchronized with microwave pulses. Optical EPR techniques that are on the forefront of magnetic resonance research can be used to study photochemical reactions and light-induced excited states of flavin sites in bifurcating enzymes.
Bifurcating enzymes are complex and contain multiple FeS and flavin cofactor sites. To help address the challenge of understanding how these function in electron bifurcation and transfer reactions, the ASRS is equipped with the latest in pulse-EPR technology. Pulse EPR is a high-resolution technique, making it possible to resolve multiple components and connect electronic structure to molecular structure. Pulse capabilities including ENDOR and HYSCORE will provide approaches to determine local structure surrounding spin centers. The techniques probe electron-nuclear interactions and open up new possibilities to characterize reaction intermediates and unique cofactor environments.
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Cara Lubner promoted to staff scientist at NREL
Cara Lubner joined NREL and BETCy as a postdoc in the fall of 2014 and recently converted to a full time staff scientist. She works in Paul King's research group at NREL. She previously studied photosynthetic electron transfer and the catalytic production of hydrogen in
John Golbeck's lab at
Penn State. For the past year, Cara has been probing flavin intermediate states in several flavoproteins and bifurcating enzymes using ultrafast transient absorption spectroscopy (TAS), together with other biophysical techniques. As she continues to work at NREL as a member of BETCy, Cara looks forward to applying her expertise in ultrafast laser spectroscopy towards uncovering the mysteries of electron bifurcation.
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