Grants and Contracts Details
Description
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.
Status | Active |
---|---|
Effective start/end date | 9/1/20 → 8/31/25 |
Funding
- Department of Energy: $725,274.00
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