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.
more »
« less
This content will become publicly available on May 22, 2025
A single hydrogen bond that tunes flavin redox reactivity and activates it for modification
Electron bifurcation produces high-energy products based on less energetic reagents. This feat enables biological systems to exploit abundant mediocre fuel to drive vital but demanding reactions, including nitrogen fixation and CO2 capture. Thus there is great interest in understanding principles that can be portable to man-made devices. Bifurcating electron transfer flavoproteins (Bf ETFs) employ two flavins with contrasting reactivities to acquire pairs of electrons from the modest reductant, NADH. The flavins then disperse the electrons individually to high- and a low reduction midpoint potential (E°) acceptors, the latter of which captures most of the energy. Maximum efficiency requires that only one electron access the exergonic path that will 'pay for' production of the low-E° product. It is therefore critical that one of the flavins, the 'electron transfer' (ET) flavin, is tuned to execute single-electron (1e—) chemistry only. To learn how, and extract fundamental principles, we systematically altered interactions with the flavin O2 position. Removal of a single hydrogen bond (H-bond) disfavored formation of the flavin anionic semiquinone (ASQ) relative to the oxidized (OX) state, lowering E°ASQ/OX by 150 mV and retuning the flavin's tendency to 1e— vs. 2e— reactivity. This was achieved by replacing conserved His 290 with Phe while also replacing the supporting Tyr 279 with Ile. Although this variant binds oxidized FADs at 90% the WT level, the ASQ state of the ET flavin is not stable in the absence of H290's H-bond, and dissociates, contrary to WT. Removal of this H-bond also altered the ET flavin's covalent chemistry. Whereas the WT ETF accumulates modified flavins whose formation is believed to rely on an anionic paraquinone methide intermediate, the FADs of the H-bond lacking variant remain unchanged over weeks. Hence the variant that destabilizes the anionic semiquinone also suppresses the anionic intermediate in flavin modification, testifying to electronic similarities between these two species. These correlations suggest that the H-bond that stabilizes the crucial flavin ASQ also promotes flavin modification. The two effects may indeed be inseparable, as a Jekyll and Hydrogen bond.
more »
« less
- Award ID(s):
- 2108134
- PAR ID:
- 10556072
- Publisher / Repository:
- Royal Society of Chemistry
- Date Published:
- Journal Name:
- Chemical Science
- Volume:
- 15
- Issue:
- 20
- ISSN:
- 2041-6520
- Page Range / eLocation ID:
- 7610 to 7622
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Recently, a variety of enzymes have been found to accept electrons from NAD(P)H yet reduce lower-potential carriers such as ferredoxin and flavodoxin semiquinone, in apparent violation of thermodynamics. The reaction is favorable overall, however, because these enzymes couple the foregoing endergonic one-electron transfer to exergonic transfer of the other electron from each NAD(P)H, in a process called 'flavin-based electron bifurcation'. The reduction midpoint potentials (E°s) of the multiple flavins in these enzymes are critical to their mechanisms. We describe methods we have found to be useful for measuring each of the E°s of each of the flavins in bifurcating electron transfer flavoproteins.more » « less
-
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.more » « less
-
more » « less
Abstract Flavins have emerged as central to electron bifurcation, signaling, and countless enzymatic reactions. In bifurcation, two electrons acquired as a pair are separated in coupled transfers wherein the energy of both is concentrated on one of the two. This enables organisms to drive demanding reactions based on abundant low‐grade chemical fuel. To enable incorporation of this and other flavin capabilities into designed materials and devices, it is essential to understand fundamental principles of flavin electronic structure that make flavins so reactive and tunable by interactions with protein. Emerging computational tools can now replicate spectra of flavins and are gaining capacity to explain reactivity at atomistic resolution, based on electronic structures. Such fundamental understanding can moreover be transferrable to other chemical systems. A variety of computational innovations have been critical in reproducing experimental properties of flavins including their electronic spectra, vibrational signatures, and nuclear magnetic resonance (NMR) chemical shifts. A computational toolbox for understanding flavin reactivity moreover must be able to treat all five oxidation and protonation states, in addition to excited states that participate in flavoprotein's light‐driven reactions. Therefore, we compare emerging hybrid strategies and their successes in replicating effects of hydrogen bonding, the surrounding dielectric, and local electrostatics. These contribute to the protein's ability to modulate flavin reactivity, so we conclude with a survey of methods for incorporating the effects of the protein residues explicitly, as well as local dynamics. Computation is poised to elucidate the factors that affect a bound flavin's ability to mediate stunningly diverse reactions, and make life possible.
This article is categorized under:
Structure and Mechanism > Computational Biochemistry and Biophysics
Electronic Structure Theory > Combined QM/MM Methods
Theoretical and Physical Chemistry > Spectroscopy
-
Schepartz, Alanna (Ed.)The physiological role of dihydroorotate dehydrogenase (DHOD) enzymes is to catalyze the oxidation of dihydroorotate to orotate in pyrimidine biosynthesis. DHOD enzymes are structurally diverse existing as both soluble and membrane-associated forms. The Family 1 enzymes are soluble and act either as conventional single subunit flavin-dependent dehydrogenases known as Class 1A (DHODA) or as unusual heterodimeric enzymes known as Class 1B (DHODB). DHODBs possess two active sites separated by ∼20 Å, each with a noncovalently bound flavin cofactor. NAD is thought to interact at the FAD containing site, and the pyrimidine substrate is known to bind at the FMN containing site. At the approximate center of the protein is a single Fe2S2 center that is assumed to act as a conduit, facilitating one-electron transfers between the flavins. We present anaerobic transient state analysis of a DHODB enzyme from Lactoccocus lactis. The data presented primarily report the exothermic reaction that reduces orotate to dihydroorotate. The reductive half reaction reveals rapid two-electron reduction that is followed by the accumulation of a four-electron reduced state when NADH is added in excess, suggesting that the initial two electrons acquired reside on the FMN cofactor. Concomitant with the first reduction is the accumulation of a long-wavelength absorption feature consistent with the blue form of a flavin semiquinone. Spectral deconvolution and fitting to a model that includes reversibility for the second electron transfer reveals equilibrium accumulation of a flavin bisemiquinone state that has features of both red and blue semiquinones. Single turnover reactions with limiting NADH and excess orotate reveal that the flavin bisemiquinone accumulates with reduction of the enzyme by NADH and decays with reduction of the pyrimidine substrate, establishing the bisemiquinone as a fractional state of the two-electron reduced intermediate observed.more » « less
