Author ORCID Identifier

Date Available


Year of Publication


Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation


Arts and Sciences



First Advisor

Dr. Anne-Frances Miller


Electron bifurcation is considered as a third fundamental mode of energy conservation mechanism in addition to two well-known mechanisms, substrate level phosphorylation and Oxidative phosphorylation, in electron bifurcation endergonic and exergonic redox reactions are coupled. The newly discovered flavin based electron bifurcation in electron transfer flavoproteins (ETFs) helps to reduce low potential ferredoxin, which provides electrons to drive biologically demanding reactions such as atmospheric dinitrogen fixation in diazotroph and methane production in methanogens.

Current research demonstrates the capacity for electron bifurcation in the Rhodopseudomonas palustris ETF (RpalETF) system. RpalETF contains two chemically identical but functionally different FADs: ETFAD is bound in highly mobile domain II, which sits in a stable base created by domains I and III. Bf-FAD is buried in between domains I and III. The two flavins execute contrasting, complementary electron transfer reactions. Whereas one mediates single electron transfer (ET-FAD), the other accepts electrons pairwise (Bf-FAD), yet both flavins’ sites include a conserved Arg sidechain. R273 favors the anionic semiquinone (ASQ) of ET-FAD, whereas R165 near the Bf-FAD appears not to, possibly due to neutralization of its positive charge by nearby C174. R273 forms a 𝜋-𝜋 stacking interaction with ET-FAD whereas R165 appears to form hydrogen bond interactions with Bf-FAD.

To learn whether the active site arginine residues each have different effects on their respective neighboring flavins, we replaced each of the Args in turn with chemically conservative, and divergent substitutions. We utilized site directed mutagenesis to specifically replace the residues with Gln (nearly isosteric to Arg and polar), Lys (positively charged like Arg but lacking a bidentate H-bonding capability), His (having a pKa closer to 7 so that its charge is tunable by solution pH), and Ala (a nonpolar small side chain that serves as a control).Our data show, R273 plays a vital role in Bf-ETF by stabilizing the ASQ of the ET-FAD, whereas R165 favors binding of the Bf-FAD that is essential for electron bifurcation in RpalETF.

Along with the electron bifurcation studies, we report an irreversible, pH dependent, site selective, enzyme mediated, anaerobic chemical modification of ET-FAD to a pink amino FAD, which opens a new perspective with which to understand the 726 nm band formed in bifurcating ETF.

Digital Object Identifier (DOI)

Funding Information

This study was supported by the Biological and Electron Transfer and Catalysis (BETCy) EFRC, an Energy Frontier Research Center funded by the US Department of Energy, Office of Science, Basic Energy Sciences under Award DE-SC0012518 from 1 Aug. 2014 - 31 July 2018 and The National Sciences Foundation, Chemistry of Life Processes CHE-1808433 from 1 July 2018 - 31 Dec 2021.