Differentiating the Two Complementary Flavins in a Bifurcating Electron Transfer Flavoprotein

Abstract for N.S.F. grant due on 16 November 2020. Title : Redox tuning in a bifurcating electron transfer flavoprotein Intellectual Merit: Electron transfer is central to energy metabolism in all kingdoms of life. Of the redox cofactors used, FMN and FAD are among the most versatile, as the flavin moiety is inherently poised between one-electron and two-electron reactivities by the tunable stability of the flavin''s semiquinone states, and the flavin''s electron transfer reactions are coupled to proton transfer. This tunability is central to the ability of electron transfer flavoproteins to mediate electron transfer bifurcation, which in turn is of fundamental basic scientific importance because it enables enzymes to produce reductants that are more reactive than the starting materials. Bifurcating ETFs do not in fact break the laws of thermodynamics. Although a stronger reductant is produced at the expense of NADH, only one of NADH''s pair of electrons goes to lower potential, whereas the other electron passes to a high-potential accesptor thereby achieving overall thermodynamic spontaneity. Requirements for bifurcating activity include one redox site at which single-electron transfers are not favoured (the semiquinone is suppressed) and another site that exclusively accepts a single electron. Paradoxically, both of these are FAD, in the bifurcating ETFs. Thus these proteins provide a single system incorporating a pair of complementary flavins, each of which serves as a built-in control for the other. We seek to understand what differences between the two sites are responsible for the different reactivities their flavins display. We also seek to understand reactivity that involves both flavins at once. In particular, a novel charge transfer species develops in ETF containing both flavins but not in ETF lacking either one. This species is correlated with formation of a covalently modified flavin, only when both flavins are present. We hypothesize that electron transfer between the two flavins or some sort of collective activity is responsible for both. Thus we will treat both as probes of how the reactivity of this BfETF is greater than the sum of its two individual flavins. To understand the forces that shape individual flavin reactivities and ways in which they function together we will (1) determine the identities of modified flavins produced in our Bf-ETF, elucidate mechanisms by which they are formed and test for a correlation with bifurcating activity and ETF conformation, (2) develop the use of computation to understand optical spectal changes in terms of flavin electronic structure and (3) identify mechanisms of redox tuning by which the protein modulates the stabilities of the flavin semiquinones, to achieve energetic coupling between the two electrons transferred. Broader Impacts: The plant pigments, fibers and fragrances course was a success for the students who participated. However due to limitations required in response to Covid-19, participation was tightly limited to maintain social distancing. To overcome this and expand this course''s ability to make chemistry appealing, and chemical concepts accessible, I propose to produce a modular text and studio manual enabling home-schoolers and rural schools throughout Kentucky to offer the plant pigments fragrances and fiber course locally and to audiences of adult learners as well as advanced high-school audiences. **I would like to consult or collaborate with Agriculture Extension. What form should this take in order to keep this grant application simple**