In this issue...
The research described in this newsletter is supported as part of the 
Biological Electron Transfer and Catalysis, an Energy Frontier Research Center funded by the   U.S. Department of Energy, Office of Science.
bifurcation
BETCy scientists publish on the thermodynamics and kinetics of electron bifurcation

In collaboration between BETCy experimentalists at Washington State University and National Renewable Energy Laboratory, theoretical chemists Peng Zhang and Jonathon Yuly in BETCy's Beratan research group at Duke University published in the September 2017 issue of Accounts of Chemical Research .
 
This paper accomplished two significant goals. First, it is one of the first publications to place biological electron bifurcation on firm thermodynamic footing. Second, it rationalizes the correct electron transfer sequence for the bifurcating steps of the catalytic cycle in the Nfn enzyme (NADH-dependent reduced ferredoxin: NADP+ oxidoreductase I).
 
In their analysis of the thermodynamics of the reaction, the authors considered the reduction potentials of the cofactors and the distances between cofactors, assuming that the initial steps along each branch obey the kinetics of nonadiabatic electron-transfer theory.  The Nfn reaction mechanism begins with sending the first electron from the bifurcating flavin site up the high potential branch towards NAD+.
 
The first electron transfer switches on a second thermodynamically spontaneous electron transfer reaction from the flavin along a second pathway that moves the second electron in the opposite direction and at a lower potential.  The second electron is sent down the low potential branch towards ferredoxin; thus the second electron to leave the flavin is much more reducing than the first: the potentials are said to be "crossed."
 
Using thermodynamic considerations, the paper shows that electron bifurcation can occur without crossed potentials. However, crossed potentials may yield distinct advantages for biological electron bifurcation. For instance, when a bifurcating species is in the crossed potential landscape, its low potential redox state can be extremely short lived (~10 ps in Nfn). This allows the system to use that highly reducing state without having to protect it from energy dissipating electron acceptors.
 
Graduate students Diep Nguyen at UGA and Jonathon Yuly at Duke presented the results of this study as part of the Team Science Competition held at the 2017 EFRC PIs Meeting in Washington, DC.
electron
BETCy Team Establishes Role of Electron Bifurcation in Nitrogen Fixation
 
The nitrogenase enzyme catalyzes the reduction of dinitrogen (N2) to a bioavailable form (NH3) in an energy intensive reaction that requires both ATP and a strong reductant containing high-energy electrons. The pathways that generate the high-energy electrons, however, have remained elusive for many nitrogen-fixing bacteria.
 
In a recent publication, BETCy scientists showed that some nitrogen-fixing bacteria generate strong reductants using the so-called FixABCX enzyme complex. The Fix complex bifurcates electrons from NADH, sending half the electrons down a high-energy pathway while sending the remaining electrons down a lower energy path.
 
Lead author Rhesa Ledbetter, a graduate student in the Seefeldt research group at Utah State University, started by purifying the electron-bifurcating FixABCX complex from the nitrogen-fixing bacterium, Azotobacter vinelandi. The purified complex was biochemically characterized, and demonstrated to generate reductant for nitrogen fixation using a mechanism that involves electron bifurcation. 

Biochemical and biophysical data supported a pathway in which two electrons are donated to FixABCX from NADH and then split to proceed down two different pathways-one electron going down the exergonic branch to coenzyme Q and the other traveling down the endergonic branch to a small electron carrier protein, flavodoxin. The high-energy electrons in flavodoxin can then be used to as the reductant for nitrogenase. The BETCy team also confirmed that the FixABCX complex does, in fact, donate electrons to nitrogenase in A. vinelandii whole cells, confirming a physiological role.
 
Ov erall, this work establishes a new pathway for the generation of reductant for one of the most difficult biological reactions and provides insight into roles of electron bifurcating complexes in living systems.
 
The work on the FixABCX electron bifurcating complex was published in the   Aug. 15, 2017 issue of the journal Biochemistry and was a collaboration among Utah State University, Montana State University, National Renewable Energy Laboratory, Idaho State University, University of Minnesota, University of Kentucky, and Washington State University.
NucleotideNucleotide-dependent structural changes drive protein-protein interactions essential for nitrognease activity 

A recent paper by BETCy scientists is providing fresh insight into the mechanisms controlling electron transfer within nitrogenase, the multi-subunit enzyme complex responsible for biological nitrogen fixation. The interdisciplinary team, led by three BETCy research groups, probed the protein-protein interactions involved in electron transfer between different components of nitrogenase.

A central component of electron transfer in nitrogenase is the so-called Fe protein cycle, an ATP-dependent process, which involves a transient association between the reduced, MgATP-bound Fe protein and the MoFe protein and includes electron transfer, ATP hydrolysis, release of Pi, and dissociation of the oxidized, MgADP-bound Fe protein from the MoFe protein. The cycle is completed by reduction of oxidized Fe protein and nucleotide exchange.


In the current work, the BETCy team focused on how the electron donor, flavodoxin, interacts with the Fe protein. Understanding how flavodoxin interacts with the Fe protein in its different nucleotide-bound conformations is important for a fundamental understanding of the Fe protein cycle. A major challenge was that while the structure of the Fe protein has been determined in the nucleotide-free and MgADP-bound state, the structure of the MgATP-bound state of the Fe protein has remained elusive.

Using time-resolved proteolysis and chemical cross-linking in combination with mass spectrometry, the BETCy team identified nucleotide-induced structural changes in the Fe protein and analyzed their effects on interactions with flavodoxin. Differences in proteolytic cleavage and cross-linking patterns were consistent with known nucleotide-induced structural differences in the Fe protein and indicated that MgATP-bound Fe protein resembles the structure of the Fe protein in the stabilized nitrogenase complex structures.

Lead author Natasha Pence, from the Peters research group said, "Confirming that we could use these two techniques to differentiate the Fe protein in its different nucleotide-bound states was the key." Cross-linking and proteolysis patterns as well as in silico docking studies demonstrated that the MgADP-bound state has the most complementary docking interface with flavodoxin.

The difference in complementarity results in less competition between the MoFe protein and flavodoxin binding to the MgADP-bound Fe protein. The results provide a deeper understanding of the Fe protein cycle and the capabilities developed for this project enables further BETCy research into the mechanisms of electron transfer utilized by other enzyme systems.

The article, " Unraveling the interactions of the physiological reductant flavodoxin with the different conformation of the Fe protein in the nitrogenase cycle" appeared in the Sept. 22, 2017 issue of the Journal of Biological Chemistry. 

flavoproteinDefining Electron Bifurcation in the Electron Transferring Flavoprotein Family

BETCy researchers have classified the diverse family of electron transferring flavoproteins (Etfs) into five distinct groups and have identified a set of amino-acid motifs that predict the ability of enzymes to catalyze electron bifurcation reactions. The new study appears in the November 2017 issue of the Journal of Bacteriology.
Etfs are a diverse group of enzymes although only a small number of these enzymes have been biochemically characterized. Importantly, crystal structures have been solved for Etfs that bifurcate and also solved for those that do not bifurcate. This provides a unique opportunity to identify the structural determinants that dictate bifurcation capability for use in identifying new bifurcating enzymes.

Graduate student Saroj Poudel from BETCy's Boyd laboratory and postdoctoral researcher Amaya Garcia-Costas from the Peters laboratory worked together to develop a database of Etf enzymes encoded in available genome sequences. In collaboration with personnel from three additional BETCy research groups, they examined this new database of sequences to identify the structural determinants that delineate the capability to bifurcate electrons.

They identified five phylogenetically and functionally coherent Etf groups. Importantly, only one group contained bifurcating Etfs. Combined structural and bioinformatics analysis of representatives of bifurcating and non-bifurcating Etfs revealed a suite of amino-acid motifs that demarcates bifurcating Etfs from non-bifurcating Etfs. Further, this suite of amino acids was used to identify unique lineages of putative bifurcating enzymes that have no defined function in host cells.

This work broadens our understanding of the diversity of bifurcating enzymes in natural systems and provides new templates for designing experiments to elucidate the mechanistic details of flavin based electron bifurcation.

Structural modeling reveals key differences between bifucating Etfs (left) and non-bifurcating Etfs (right).

Xfn
BETCy publication highlights intriguing differences between Nfn and Xfn



Building on work published earlier this year  that describes the mechanism of flavin-based electron bifurcation in the enzyme Nfn, BETCy recently published an additional paper that provides the first structural and biochemical characterization of Xfn, an enzyme which is closely related to Nfn. Both Nfn and Xfn have overlapping roles in maintaining cellular redox balance.

Nfn uses a bifurcating mechanism to exchange reducing units between the electron carriers NADPH, NADH and ferredoxin. In the new study, BETCy scientists uncover important differences between the Nfn and Xfn enzymes and provide comparisons between the two that will aid researchers to better understand the atomic determinants of enzymes that catalyze electron bifurcation.

The collaborative effort between BETCy scientists from UGA, MSU and WSU entitled " Two functionally distinct NADP+-dependent ferredoxin oxidoreductases maintain the primary redox balance of Pyrococcus furiosus" was published in the Sept. 1, 2017 issue of the Journal of Biological Chemistry.

In this paper, BETCy demonstrated for the first time the importance of the Nfn and Xfn in maintaining the electron pool of the three most ubiquitous electron carriers inside P. furiosus (ferredoxin, NAD(H) and NADP(H)). Deleting genes encoding for either Nfn or Xfn affected the cellular concentrations and ratios of NADPH/NADP, which, in turn, caused moderate to severe growth defects in mutant strains.

As part of the new work, BETCy solved the crystal structure of Xfn to complement BETCy's earlier structure of Nfn. Comparative studies between Nfn and Xfn at the structural and biochemical levels showed that despite the close similarities between the two enzymes in terms of the primary sequence, subunit composition and cofactor content, Xfn did not catalyze the same bifurcating reaction as Nfn. Instead, it exhibits a non-bifurcating ferredoxin NADP oxidoreductase-type activity.

By superposition of the two enzyme structures, researchers noted differences in the NADH binding site, which was blocked by an additional loop in the small subunit of Xfn. The additional loop in Xfn, which apparently prevents NADH binding, is intriguing and may point to the use by Xfn of an alternative substrate, the identity of which remains unknown.

This new study enhances our perspective into the active site requirements necessary for electron bifurcation in Nfn-type enzymes.
time
BETCy groups combine time-domain and equilibrium spectroscopic studies to refine diagnosis of bifurcating flavins

A new BETCy paper appearing in the Aug. 25 issue of the  Journal of Biological Chemistry builds on BETCy's pioneering transient absorption spectroscopy (TAS) studies to explain the short lifetimes of bifurcating vs. non-bifurcating flavin anionic semiquinones (ASQs) in terms of their mechanisms of re-oxidation via electron transfer vs. charge recombination.
 
TAS revealed short-lived flavin ASQs in each of the flavin-based bifurcating systems studied by BETCy. These observations provided important first evidence for a mechanism in which an unstable flavin semiquinone could transfer an electron to a low-potential acceptor. 

The new study makes sense of the fact that short-lived ASQs are not unique to bifurcating systems. Indeed the team from  University of Kentucky, National Renewable Energy Lab and  University of Georgia compared a series of bifurcating and non-bifurcating systems and documented ASQ lifetimes ranging over two orders of magnitude, with that of bifurcating transhydrogenase (Nfn) falling near the middle of the range on a log scale. Thus, while a short-lived ASQ may be necessary for bifurcation, it is clearly not sufficient to infer that bifurcation occurs.
 
To understand what properties might identify a bifurcating flavin, the researchers probed the mechanisms by which photogenerated ASQ decays. Using the slowly-decaying ASQ of nitroreductase (NR), the work exploited exogenous electron donors to modulate the contribution of charge recombination, achieving more than hundred-fold acceleration of ASQ decay to produce an ASQ lifetime as short as the shortest one observed for a natural system, and ten times shorter than that of bifurcating Nfn.
 
The authors combined absorption and fluorescence spectroscopy to demonstrate formation of charge transfer complexes between the flavin and the most efficient exogenous donors. Thus, local electron transfer from the bound exogenous donor to photoexcited flavin and then back to oxidized donor from ASQ can explain the short ASQ lifetimes produced by these donors. This contrasts with the extended series of electron carriers seen in Nfn, which propagate electron density between the flavin and other cofactors tens of angstroms away. The combination of methods permits a more nuanced interpretation of ASQ lifetime in terms of the mechanism of ASQ re-oxidation, via charge recombination vs. efficient electron transport.
 
This approach can aid BETCy researchers in identifying flavins whose short ASQ lifetimes could reflect capacity for electron bifurcation. 

This work by   BETCy scientists ranging from undergraduates through graduates students, research scientists and faculty was launched by a BETCy ETC award (BETCy's internal travel award program) which enabled BETCy graduate student  J. P. Hoben  from UK to perform the work with other BETCy scientists at NREL. BETCy's ETC program was highlighted in the  Summer 2017 issue of this newsletter.