Ancient lineages of bacteria seem able to flourish on meager resources and low quality chemical fuel. This remarkable metabolic 'super-power' has been explained in part by their use of a biochemical strategy called electron transfer bifurcation ('bifurcation'). We want to learn how this is accomplished, with the goal that humanity's abundant low-grade fuels, such as solar and wind-derived current, might thus be transformed into energy-dense forms and used instead of fossil fuels.
Bifurcating electron transfer flavoproteins (bETFs) execute bifurcation based on the activities of two flavins bound by the protein, in concert with two partner proteins. Since flavins (e.g. vitamin B2) are non-toxic fully renewable electron carriers, they are very attractive catalysts and we want to learn how bETF protein activates them for their roles. Crucially, although bETF contains two molecules of flavin adenine dinucleotide (FAD), they display very different chemical reactivities (the word 'flavin' denotes the portion of FAD that is chemically reactive). Their distinct reactivities must be attributed to different interactions with the protein in the two flavins' binding sites, since the FADs themselves are chemically identical. The FAD that executes bifurcation is called the bifurcating flavin (Bf-flavin) whereas the FAD whose flavin relays electrons from the bifurcating flavin to the partners is called the electron transfer flavin (ET-flavin). The latter's ability to transfer electrons one at a time is unusual among flavoproteins, and reflects highly sophisticated modulation of the energies of the electrons being transferred.
Our EVF-funded work employed quantum chemical calculations to characterize the interactions with the protein for each of the two flavins. We proposed why the ET-flavin is able to transfer electrons one at a time, whereas the Bf-flavin does not. Indeed, when another team-member from the Miller group tested the significance of the interaction identified computationally, she found that its removal decreased the single-electron activity of the ETflavin. We used other computational tools to simulate large molecular motions that are theorized as part of the bETF's catalytic cycle. The protein as a whole can be likened to a cup and saucer, wherein the cup must be upright in one half of the catalytic cycle but inverted on the saucer in the other. Since the protein has no external hand to flip the 'cup', we used calculations to test whether the 'cup' (or head domain) could roll back and forth (only by 80°, not a full 180°). We also tested popular hypotheses as to the nature of the trigger that initiates head rotation. Our calculations found that acquisition of electrons by the ET-flavin do not trigger head domain rotation, but that presence of a bound electron delivery molecule can. When the molecule that delivers electrons is bound near the Bf-FAD, our calculations identify new conformations of the protein that are not common in the absence of that molecule. Thus, we are learning how interactions and motions of the protein control the reactivities of the flavins that mediate bifurcation, and enable lowly bacteria to accomplish enviable feats.