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Due to the presence of O2 as electron acceptors in aerobes, the energy metabolism is 15 times more efficient than anaerobic metabolism. Krebs’s cycle and oxidative phosphorylation results in the production of additional 36 mols of ATP in aerobes.  However, it was observed in Clostridium kluyveri that during pyruvate fermentation to butyrate, H2 was generated. This mitigated energy deficit in bacteria and archaea but raised concerns as to how could mediocre fuels generate H2, a high energy reductant. This endergonic transfer violated the laws of thermodynamics suggesting the tight coupling to an exergonic reaction paying off for the endergonic transfer. This led to the discovery of Flavin based electron bifurcation (FBEB) in 2008 which could answer the mechanistic details behind the tight coupling of an exergonic pathway to an endergonic pathway leading to the production high energy fuel from mediocre ones. Thus, FBEB is considered as the third mode of energy conservation and is crucial for bacteria and archaea to carry out CO2 and N2 fixation. Bifurcating electron transfer flavoproteins (bETF) are heterodimeric flavoproteins that carry out FBEB. bETFs are comprised of two subunits- larger EtfA formed by domain I and domain II and smaller EtfB formed by domain III. Domains I and III form the base of the protein whereas domain II sits on top of the base and is known to be dynamic shuttling towards and away from the base. bETFs contain two non-covalently bound flavins- bifurcating FAD (Bf- FAD) situated at the interface of domain I and domain III and electron transfer FAD (ET-FAD) positioned in domain II. In FBEB, NADH is a natural substrate for these bETFs which donates 2 e- in the form of a hydride completely reducing the Bf-FAD. From the reduced HQBf-FAD, one e- goes downhill to the high potential acceptor (ET-FAD in this case) and the second e- flows uphill to a low potential acceptor (flavodoxin or ferredoxin). The two pathways are tightly coupled, and the overall energetics of the system is retained. Electron gating is crucial towards the mechanism of the reaction which allows the second e- to flow uphill instead of downhill in the favorable direction. Apart from the protein dynamics which prevents the flow of the second electron to the exergonic pathway, tuning ET-FAD’s 1 e- reactivity allowing it to do 1 e- chemistry unlike Bf-FAD’s 2 e- chemistry is crucial for electron gating. Flavins can accumulate up to 1% semiquinone in solution. Contrasting reactivities of the two FAD’s is highly unique in these systems as these are the polypeptide environment of the respective FAD that tunes their potential over a wide range of reactivity. It is very important to understand the properties and reactivities of these bETFs in order to be able to make it potable to mankind to produce high energy fuel from mediocre and abundant ones.  My study involves the biophysical and biochemical characterization of a thermophilic flavoprotein to establish it as a bifurcating ETF and H-bonding from a conserved histidine residue in ETFs that is responsible for tuning ET-FAD’s 1 e- reactivity and  unusual formation of 8-amino flavin in the ET-site.