KY EPSCoR: FY21 & FY22: Match to 3200003615 - Gating Electron Transfer in Biological Energy Storage and Conversion

Grants and Contracts Details


Electron transfer flavoproteins (ETFs) accept electrons from enzymes that extract them from fuel molecules, and deliver them to enzymes employing them to do work. A family of ETFs has been discovered that can execute electron transfer bifurcation. The ETF acquires electron pairs at intermediate potential but delivers them in separate energetic directions, producing one electron at a much more reducing potential able to drive nitrogen fixation, whilst the other electron is released at more weakly reducing potential (conserving total energy). Thus, bifurcation allows biochemical systems to use cheap abundant fuel to drive critical demanding reactions. Bifurcating ETFs (Bf-ETFs) are therefore important targets for DOE research. To enable manmade materials and micro-electronic devices with Bf ability, we are elucidating fundamental requirements for bifurcation. The Bf-ETFs we study support nitrogen fixation, another DOE priority, and the project addresses conformational changes believed to gate electron transfer, another important theme in DOE-funded bio-energetics. There is as-yet no unified understanding of the bases for Bf-ETFs'' abilities to direct individual electrons to different acceptors at different energies. With two paths available, the ETF must be able to sever the energetically down-hill path to ensure that high-energy electrons do not exploit it, yet the down-hill path must carry off low-energy electrons to satisfy thermodynamics. A gating mechanism coordinated with elements of catalysis is thus crucial. The Small Angle Neutron Scattering (SANS) capability of Oak Ridge National Laboratory (ORNL) is a perfect match for this research question as SANS can detect conformational change but does not perturb oxidation states of flavins, unlike more commonly-used X-ray or FRET methods. The head domain of ETFs undergoes a = 80° rotation that carries the electron transfer (ET) flavin back and forth between a position close to the bifurcating (Bf) flavin to a position exposed to e- accepting partners. This is the proposed conformational gate that could allow vs. prevent electron transfer between the Bf and ET flavins. -1- To learn which catalytic events are tied to (and could trigger) the domain movement, we will use SANS to compare samples prepared in different oxidation states proposed to represent intermediates of the catalytic cycle. Similarly, to learn whether substrate binding / product release is coupled to conformation change we will compare ETF ± inert NADH/NAD+ analogs. To maximize the discriminating power of SANS, we will employ subunit-selectively deuterated ETF (Large subunit ''L''deuterated with Small subunit ''S'' proteated, or vice versa). This takes advantage of the very different scattering of neutrons by 1H vs, 2H that can allow us to deconvolute contributions from each of the two subunits of ETF. The project will moreover develop new capability to handle air-sensitive samples at the SANS instrumentation at ORNL that will permit spectrophotometric monitoring of the redox state of the enzyme. 2- To learn whether binding of partner proteins trigger the conformational change, we will express and purify the partner protein FixX. Exploiting another SANS strategy, we will experiment with partial deuteration of FixX to identify an isotope ratio that results in net-zero neutron scattering. In aim 3 we will use the ''invisible'' FixX, collecting SANS data on complexes of it with isotopomers of the ETF (2L•1S or 1L 2S) wherein net scattering will reflect only the ETF, enabling us to compare the conformations of ETF ± FixX. Graduate students from the Miller lab will generate ETF and FixX at the University of Kentucky or at ORNL''s biodeuteration facility, each spending a semester at ORNL (one student per year) to learn biodeuteration techniques, collect SANS data, and employ computational modelling to interpret the experimental data, all under the guidance of ORNL experts.
Effective start/end date9/1/206/30/23


  • KY Economic Development Cab: $50,000.00


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