Anaerobic bacteria and archaea thrive in seemingly inhospitable environments because they are extremely energy e铿僣ient. Their e铿僣iency is based in large part on their ability to conduct electron transfer bifurcation ('bifurcation') at strongly reducing potentials, thereby producing extremely potent reducing agents able to 铿亁 nitrogen and make molecular hydrogen. This chemistry is made possible by the use of a 铿俛vin as the site of bifurcation, supported by a specialized protein environment and mechanisms that control the 铿俹w of individual electrons.
Bifurcating electron transfer 铿俛voproteins (Bf-ETFs) are versatile protein modules that provide the bifurcating capability associated with several metabolic functions. Bf-ETFs enable use of low-energy electron reserves such as NADH to charge the carriers ferredoxin and 铿俛vodoxin with high-energy electrons. Bf-ETFs possess two 铿俛vin adenine dinucleotide (FAD) cofactors. The bifurcating FAD (Bf-FAD) receives two electrons from NADH, and distributes them through two distinct pathways. One pathway involves exothermic electron transfer to a high- potential acceptor via the second FAD, the ET-FAD (electron transfer FAD). This provides the driving force to send the second electron to a lower potential (higher-energy) acceptor.
Investigations described herein elucidated the crystal structure and internal dynamics of 铿俛vodoxin (Fld), a high-energy acceptor in the bifurcation process. 19F NMR was used to examine conformational heterogeneity and dynamics of Fld free in solution, to characterize the 铿俥xibility of a 20-residue stretch of Fld's peptide chain that is believed to mediate interaction between Fld and ETF. Temperature-dependent NMR studies, alongside paramagnetic relaxation investigations comparing Fld in both its oxidized and semi-reduced forms, detailed internal dynamics pivotal to Fld's interactions with diverse partner proteins.
Complementary research explored conformational dynamics of ETF, employing small-angle neutron scattering (SANS). This revealed notable divergence from published structures, demonstrating presence of a more extended conformation in solution. Signi铿乧ant reduction- triggered conformational change was also discerned via SANS by comparing the fully oxidized and reduced states of ETF. Molecular dynamics simulations-based data modeling suggests coexistence of multiple ETF conformations, ranging from extended to compact, in solution.
Finally, conformational consequences of complex formation between ETF and a partner protein were examined. We demonstrated isolation of a complex between ETF and its high- potential acceptor butyryl CoA dehydrogenase (BCD). Innovative application of segmental deuteration of BCD in combination with SANS, enabled comprehensive insights into the conformational adaptations made by ETF upon complex formation. Contrast variation SANS, utilizing 80% deuterated BCD, was used to identify the match point, paving the way for advanced analysis of the complex's structural dynamics.
This work enriches comprehension of the roles played by dynamics in bifurcation, and advances new technical approaches for future explorations of conformational changes within multidomain proteins.