In absence of O2 as terminal electron acceptors in anaerobic bacteria and archaea, carbohydrate metabolism is 15 times lesser efficient compared to aerobic energy metabolism resulting in energy deficit conditions. Despite their meager resources these anaerobes, they were able to generate H2 and a chemiosmotic potential able to drive energy demanding reactions such as CO2 or N2 fixation. These observations raised concerns as production of high energy reductants (H2) from mediocre fuels (NADH) defied the laws of thermodynamics.
In 2008, a known mechanism 鈥渆lectron bifurcation鈥 but with flavins as redox mediators instead of quinones was able to overcome the thermodynamic problems behind the machinery for H2 production and was termed as flavin based electron bifurcation (FBEB). FBEB couples an energetically uphill electron transfer to a downhill electron transfer, making the process favorable overall while generating high energy reductants from mediocre and abundant fuel.
A relatively simply system that exemplifies FBEB is the bifurcating electron transfer flavoproteins (bETF). bETFs are usually heterodimeric flavoproteins 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) is situated at the interface of domain I and domain III and electron transfer FAD (ET-FAD) is positioned in domain II. Although the two flavins are chemically identical, they demonstrate contrasting reactivities to facilitate an efficient electron bifurcation.
Thus, it is very crucial to understand the molecular basis of this mechanism implemented by these systems (bETFs) naturally which could be applied to man-made devices to satisfy their high energy needs.
Electron gating is a must to facilitate the mechanism which allows only one electron to access the exergonic pathway forcing the second electron to flow in the uphill direction, the major crux of the FBEB mechanism. A conformational gate has been proposed, to enforce this, but differential redox tuning of the two flavins is also required. The polypeptide environment of these bETFs tune the reactivities of the two flavins via non-covalent interactions thus conferring them contrasting reactivities : ET-FAD carries out 1 electron chemistry whereas Bf-FAD does 2 electron chemistry enabling it to capture maximum reducing power from NADH. Free flavins in solution can accumulate up to 1% semiquinone in solution when [OX]=[HQ]. Thus, it is very unique how nature facilitates ways to an efficient mechanism.
These bETFs share several conserved reactions in the ET site that stabilizes the ASQ (anionic semiquinone) state of ET-FAD. The unusually high Eo(OX/ASQ) of ET-FAD has been attributed in part to a 99% conserved Arg and a 100 % conserved Ser or Thr. However, replacement of these does not suffice to suppress the ASQ of the ET-FAD, indicating that the site employs additional interaction(s) as well.
This thesis demonstrates that a conserved His (H290 in bETF from Acidaminococcus fermentans) is critical, for the stability of ET-FADASQ. Variants of bETF in which H290 was replaced demonstrated lower accumulation of ET-FADASQ and perturbation of ET- flavin鈥檚 E辞鈥Ox/SQ by 150 mV and E辞鈥SQ/HQ by 100 mV. Additionally, we demonstrated that the non-covalent interactions responsible for stabilizing the one electron reactivity of ET-FAD is also responsible for activating the methide intermediate responsible for covalent modification of ET-FAD in these systems.
In this study we have also biochemically characterized a monomeric ETF from a thermophilic archaeon Sulfolobus acidocaldarius showing that it qualifies as a bETF. The SaETF retains optical features unique to reported bETFs drawing attention to similar flavin environments, a must for redox tuning. Moreover, via UV-vis spectroscopy and spectroelectrochemistry we were able to demonstrate the contrasting reactivities of the two flavins.
SaETF model demonstrates conservation of residues in the ET site responsible for modulation of one electron reactivity of ET-FAD in the established heterodimeric ETFs, and an ETEo(OX/ASQ) of -21 mV confirms the stabilization of ETASQ. Finally, SaETF even replicates the side effect of ASQ stabilization that is seen in established ETFs, that the ET-FAD of SaETF is prone to covalent modification. Thus, in ongoing work, we have documented the formation of different covalently modified FADs, showing that the aerobic/anaerobic nature of the atmosphere dictates products formed, and reflected on the potent nucleophile and the reaction mechanism that allows us to refine our prior proposals for the mechanism of flavin modification.
