Conformational Controls and Consequences of Electron Transfer in Bifurcating Electron Transfer Flavoproteins

We propose to elucidate fundamental principles underlying the highly efficient energy transduction activity of bifurcating electron transfer flavoproteins, with the intent that these insights can be implemented in the production of improved man-made materials and devices for capturing, upgrading and storing energy from photovoltaics and wind sources. Electron transfer bifurcation (''bifurcation'') uses an exergonic electron transfer to drive an endergonic electron transfer, thereby producing one increased-potency reductant per pair of electrons consumed. This energy upgrade (for half the electrons) enables life to use abundant low-grade fuels to drive such vital but demanding reactions as fixation of N2 or CO2. To learn how this is accomplished, we are studying bifurcating electron transfer flavoproteins (bETFs). Exergonic electron transfer (ET) from the 2e- reduced bifurcating flavin (Bf-flavin) to a higher-E° ''ET-flavin'' (electron transfer flavin) must occur, to provide the driving force for bifurcation. However only one of each pair of electrons acquired at the Bf-flavin can be allowed to use the exergonic path. A conformational gate is believed to prevent the second electron from following the first. This 80 ° domain rotation separates the ET- flavin from the Bf-flavin, restricting the second electron to the energy conserving path to ferredoxin or flavodoxin at low potential. The endpoints of this conformational event are known from crystallography. However, solution studies are necessary to understand the motions needed to interconvert the closed conformation that supports internal ET and the open conformation that donates an electron to low-E° accceptor. In the past year, small-angle X-ray scattering (SAXS) has been published showing that the conformation of bETF is responsive to degree of reduction. However the X-rays used tend to photoreduce flavins. Indeed our recent small-angle neutron scattering (SANS) study suggests that fully oxidized ETF populates highly extended conformations in solution that are suppressed upon flavin reduction, but could enable rapid interconversion of the two compact states captured by crystallography and cryoEM. In parallel, we have developed NMR probes of protein conformation that will permit monitoring of both conformational status and degree of extension under turnover like conditions. Finally, we are developing a suite of bETF variants predicted to populate open, closed and extended conformations to different degrees. Thus, we propose to 1. Test the role of partner protein in altering the equilibrium between extended and compact states, via SANS in combination with selective isotopic labeling, and elucidate the driving forces of the equilibrium by characterizing its dependence on chaotropes, ionic strength and temperature. 2. Develop convenient 19F NMR probes to quantify population of extended vs. compact conformations via diffusion measurements, and open vs. closed via solvent exposure. In addition to modulating equilibria via solution conditions, we will compare protein variants to test roles of specific residues in stabilizing individual conformations. 3. Measure electrocatalytic activity of bETF immobilized on electrodes to test conditions for replacing one or the other of the 1e partners of bETF with the electrical circuit, and reduce NAD+, in an on-electrode reproduction of bifurcation in reverse (confurcation) Test the ability of illumination to enhance production of NADH, providing a renewable way to capture the energy of light in a chemical fuel. Thus we will elucidate determinants of bETF''s conformational ensemble, and begin developing the opportunities presented by bETF''s electrochemical activity on electrodes.