Mechanisms of Energy Conservation in Bifurcating Electron Transfer Flavoproteins

University Of Kentucky Research Foundation, Lexington KY

Investigators

Abstract

Just as electrical wires carry power to every room in our houses, cells have dedicated proteins carrying a current of electrons from reactions that generate electrons to vital reactions that require them. This project addresses a newly-recognized class of 'electron transferring flavoproteins' (Etfs) that act as energy brokers, trading quantity for quality by accepting pairs of modest-energy electrons and concentrating their energy onto just one of the pair to produce one high-energy electron. Crucially, this biochemical 'step-up station' makes it possible for cells to fix nitrogen gas from air to generate their own fertilizer, making food production possible where it otherwise would not be. The project seeks to understand HOW these Etfs accomplish this complex process. The research seeks to learn what is special about the site at which energy is reallocated among the two electrons by studying the molecule on which this happens (this flavin is a derivative of the riboflavin vitamin B2). The research also seeks to identify safety mechanisms built into the protein scaffold that allow such demanding transformations to occur within a benign, non-toxic protein. Thus the work aims to articulate the underlying principles of the process, so that they can be designed into human-made devices and materials, to increase our ability to use solar power and boost the efficiency and versatility with which we use electrical energy in general. Flavins are just one example of many plant pigments that present powerful chemical properties along with glorious colors. The project scientists share their enthusiasm for pigments via a series of workshops and a course on plant pigments, fibers and fragrances in which participants create a fiber art project using natural materials while learning about the chemical principles underlying plant colors. This creative chemistry course gives all participants a chance to design experiments and integrate science and art. A long-familiar family of proteins has recently been found to have a surprising capacity to re-allocate energy among pairs of electrons acquired from NADH, yielding one with more reducing power than the NADH source (electron transfer bifurcation, or 'bifurcation'). This research establishes which of the two flavins plays which of the contrasting roles identified in the newly-purified bifurcating electron transferring flavoprotein (Etf), and then seeks to understand what aspects of the protein environment are responsible for each of the different activities (conventional electron transfer vs. bifurcation). Spectroelectrochemical titrations measure the extent to which individual amino acids change the flavin reduction potentials (Eo) in proteins bearing amino acid substitutions, and spectroscopic studies elucidate changes in flavin covalent bonding, hydrogen bonding and protonation states, mapping out the active site's role in producing bifurcating activity. Residues to be targeted include a cysteine common to all the Etfs associated with nitrogen fixation but not conserved in others. Thus the research may reveal the protein context needed in order to exploit flavins' capacity for this reactivity while suppressing side-effects. The objective is to elucidate principles underlying the versatility and efficiency of bifurcation, for implementation beyond biochemistry. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.