Mechanisms of Energy Conservation in Bifurcating Electron Transfer Flavoproteins

1.0 Premise Energy is at the heart of life, as well as our society. Mechanisms for energy deployment that build in efficiency and conservation can play an important role in helping us achieve high quality of human life with lightened cost to the ecosystems that support us. The recently-appreciated process of electron bifurcation has been discovered in a class of electron transferring flavoproteins (ETFs) that possess a second FAD in place of the canonical structural AMP, and this additional FAD enables electron acceptance from NADH. We have purified the first such Fix-associated ETF replete with both FADs, and determined the E°s of each of the flavins. One has E°s compatible with single electron transfer (ET) whereas the other has a single 2-electron E° compatible with bifurcating (Bf) activity. The simplest interpretation of our data and that in the literature is that the 1-e FAD corresponds to the one also present in canonical ETFs and the 2-e FAD is the one that replaces the AMP of canonical ETFs and confers bifurcating activity. However this remains an assumption, unproven by direct experiment. The proposed bifurcating site contains a Cys residue that 100% conserved in diazotrophs but not other bifurcators, suggesting possible covalent association with the Bf-flavin that might have physiological regulatory significance since the Bf-FAD undergoes deleterious reactions in the presence of O2. Finally, a second electron acceptor is required if electrons are to take two paths ('bifurcate') from the Bf-FAD, but studies so far have now been able to include the physiological second acceptor FixX and test the possibility that its binding will act as a gate on steps of full bifurcating turnover. The published crystal structures set the stage for understanding bifurcating activity in terms of the different activities of the two FADs of Bf-ETFs, but in order to move intelligently on their suggestions we need to address the following questions: 1.1 Specific Aims 1. Establish which of the two flavins in the structure is the site of bifurcation vs. which one is the site of single-electron transfer. Thereby identify the protein environments that characterize each activity. 2. Determine the effect on Bf-flavin of the nearby conserved Cys174 including whether the active form of the enzyme includes a covalent flavin adduct and if so whether it might reflect a mechanism of physiological regulation. 3. Obtain a version of ETF-X competent as a low-potential electron acceptor to ETF-AB (locate the site of interaction with ETF-AB and assess possible coupling with other sites). 4. Make pigments, redox reactions (and chemistry in general) exciting and appealing to a broad audience via a hands-on course on plant pigments, fibers and fragrances offered to non-science majors at the University of Kentucky and work-shops offered to the public via the Living Arts and Sciences Center in Lexington. 1.2 Implications and Trajectory We will exploit the recent discovery of a new family of ETFs as a compact system in which to understand how nature differentially entrains a single pigment to different reactivities, and combines two instances of it to acquire qualitatively new activity: electron transfer bifurcation. Bifurcating activity has far-reaching implications and applications in production of new materials and devices that would minimize energy wastage while increasing versatility. The proposed studies will inform on fundamental properties that systems must incorporate in order to realize these possibilities, including the E°s of the individual flavins, coupling between them, association with a second electron acceptor and possible regulation of the bifurcating site in accordance with physiological conditions. The proposed work also exploits biological pigments as catalysts for discovery and learning. Color has long captivated human interest and plant pigments are embedded in the artisanal culture of Appalachian Kentucky. Therefore we will develop hands-on folk-art projects employing plant pigments, fragrances and fibers to articulate to non-scientists how the properties of molecules give rise to the natures of material. Participants will discover chemical concepts such as the greater volatility of small and non-polar molecules, the association of visible color with multiple conjugated rings or transition metal ions, and associations between 'like' molecules to cause dyes to be permanent. They will learn that