Flavin-based electron bifurcation allows enzymes to redistribute energy among electrons by coupling endergonic and exergonic electron transfer reactions. Diverse bifurcating enzymes employ a two-flavin electron transfer flavoprotein (ETF) that accepts hydride from NADH at a flavin (the so-called bifurcating FAD, Bf-FAD). The Bf-FAD passes one electron exergonically to a second flavin thereby assuming a reactive semiquinone state able to reduce ferredoxin or flavodoxin semiquinone. The flavin that accepts one electron and passes it on via exergonic electron transfer is known as the electron transfer FAD (ET-FAD) and is believed to correspond to the single FAD present in canonical ETFs, in domain II. The Bf-FAD is believed to be the one that is unique to bifurcating ETFs, bound between domains I and III. This very reasonable model has yet to be challenged experimentally. Herein we used site-directed mutagenesis to disrupt FAD binding to the presumed Bf site between domains I and III, in the Bf-ETF from Rhodopseudomonas palustris ( Rpa ETF). The resulting protein contained only 0.80 ± 0.05 FAD, plus 1.21 ± 0.04 bound AMP as in canonical ETFs. The flavin was not subject to reduction by NADH, confirming absence of Bf-FAD. The retained FAD displayed visible circular dichroism (CD) similar to that of the ET-FAD of Rpa ETF. Likewise, the mutant underwent two sequential one-electron reductions forming and then consuming anionic semiquinone, reproducing the reactivity of the ET-FAD. These data confirm that the retained FAD in domain II corresponds the ET-FAD. Quantum chemical calculations of the absorbance and CD spectra of each of WT Rpa ETF's two flavins reproduced the observed differences between their CD and absorbance signatures. The calculations for the flavin bound in domain II agreed better with the spectra of the ET-flavin, and those calculated based on the flavin between domains I and III agreed better with spectra of the Bf-flavin. Thus calculations independently confirm the locations of each flavin. We conclude that the site in domain II harbours the ET-FAD whereas the mutated site between domains I and III is the Bf-FAD site, confirming the accepted model by two different tests.
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This content will become publicly available on June 1, 2024
Noncovalent interactions that tune the reactivities of the flavins in bifurcating electron transferring flavoprotein
Bifurcating electron transferring flavoproteins (Bf-ETFs) tune chemically identical
flavins to two contrasting roles. To understand how, we used hybrid quantum mechanical
molecular mechanical calculations to characterize non-covalent interactions applied to
each flavin by the protein. Our computations replicated the differences between the
reactivities of the flavins: the electron transferring flavin (ETflavin) was calculated to
stabilize anionic semiquinone (ASQ) as needed to execute its single-electron transfers,
whereas the Bf flavin (Bfflavin) was found to disfavor the ASQ state more than does free
flavin and to be less susceptible to reduction. The stability of ETflavin ASQ was attributed
in part to H-bond donation to the flavin O2 from a nearby His side chain, via comparison
of models employing different tautomers of His. This H-bond between O2 and the ET
site was uniquely strong in the ASQ state, whereas reduction of ETflavin to the anionic
hydroquinone (AHQ) was associated with side chain reorientation, backbone
displacement and reorganization of its H-bond network including a Tyr from the other
domain and subunit of the ETF. The Bf site was less responsive overall, but formation of
the Bfflavin AHQ allowed a nearby Arg side chain to adopt an alternative rotamer that can
H-bond to the Bfflavin O4. This would stabilize the anionic Bfflavin and rationalize effects
of mutation at this position. Thus, our computations provide insights on states and
conformations that have not been possible to characterize experimentally, offering
explanations for observed residue conservation and raising possibilities that can now be
tested.
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- Award ID(s):
- 2108134
- NSF-PAR ID:
- 10478722
- Publisher / Repository:
- Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology
- Date Published:
- Journal Name:
- Journal of Biological Chemistry
- Volume:
- 299
- Issue:
- 6
- ISSN:
- 0021-9258
- Page Range / eLocation ID:
- 104762
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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