Browsing by Author "Fixen, Kathryn R."

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Defining Electron Bifurcation in the Electron Transferring Flavoprotein Family
(2017-11) Garcia Costas, Amaya M.; Poudel, Saroj; Miller, Anne-Frances; Schut, Gerrit J.; Ledbetter, Rhesa N.; Fixen, Kathryn R.; Seefeldt, Lance C.; Adams, Michael W. W.; Harwood, Caroline S.; Boyd, Eric S.; Peters, John W.
Electron bifurcation is the coupling of exergonic and endergonic redox reactions to simultaneously generate (or utilize) low and high potential electrons. It is the third recognized form of energy conservation in biology and has recently been described in select electron transferring flavoproteins (Etfs). Etfs are flavin-containing heterodimers best known for donating electrons derived from fatty acid and amino acid oxidation to an electron transfer respiratory chain via ETF quinone oxidoreductase. Canonical examples contain a flavin adenine dinucleotide (FAD) that is involved in electron transfer as well as a non-redox active adenosine monophosphate (AMP). However, Etfs demonstrated to bifurcate electrons contain a second FAD in place of the AMP. To expand our understanding of the functional variety and metabolic significance of Etfs and to identify amino acid sequence motifs that potentially enable electron bifurcation, we compiled 1,314 Etf protein sequences from genome sequence databases and subjected them to informatics and structural analyses. Etfs were identified in diverse archaea and bacteria, and these clustered into five distinct well-supported groups based on amino acid sequences. Gene neighborhood analyses indicate that these Etf group designations largely correspond to putative differences in functionality. Etfs with the demonstrated ability to bifurcate were found to form one group, suggesting distinct and conserved amino acid sequence motifs enable this capability. Indeed, structural modeling and sequence alignments revealed that identifying residues occur in the NADH and FAD-binding regions of bifurcating Etfs. Collectively, a new classification scheme is presented for Etf proteins that demarcates putative bifurcating vs. non-bifurcating members and suggests that Etf mediated bifurcation is associated with surprisingly diverse enzymes.IMPORTANCE Electron bifurcation has recently been recognized as an electron transfer mechanism used by microorganisms to maximize energy conservation. Bifurcating enzymes couple thermodynamically unfavorable reactions with thermodynamically favorable reactions in an overall spontaneous process. Here we show that the electron transferring flavoprotein (Etf) enzyme family exhibits far greater diversity than previously recognized and we provide a phylogenetic analysis that clearly delineates bifurcating and non-bifurcating members of this family. Structural modeling of proteins within these groups reveals key differences between the bifurcating and non-bifurcating Etfs.
Electron transfer to nitrogenase in different genomic and metabolic backgrounds
(2018-02) Poudel, Saroj; Colman, Daniel R.; Fixen, Kathryn R.; Ledbetter, Rhesa N.; Zheng, Yanning; Pence, Natasha; Seefeldt, Lance C.; Peters, John W.; Hardwood, Caroline S.; Boyd, Eric S.
Nitrogenase catalyzes the reduction of dinitrogen (N2) using low potential electrons from ferredoxin (Fd) or flavodoxin (Fld) through an ATP dependent process. Since its emergence in an anaerobic chemoautotroph, this oxygen (O2) sensitive enzyme complex has evolved to operate in a variety of genomic and metabolic backgrounds including those of aerobes, anaerobes, chemotrophs, and phototrophs. However, whether pathways of electron delivery to nitrogenase are influenced by these different metabolic backgrounds is not well understood. Here, we report the distribution of homologs of Fds, Flds, and Fd/Fld-reducing enzymes in 359 genomes of putative N2 fixers (diazotrophs). Six distinct lineages of nitrogenase were identified and their distributions largely corresponded to differences in the host cells' ability to integrate O2 or light into energy metabolism. Predicted pathways of electron transfer to nitrogenase in aerobes, facultative anaerobes, and phototrophs varied from those in anaerobes at the level of Fds/Flds used to reduce nitrogenase, the enzymes that generate reduced Fds/Flds, and the putative substrates of these enzymes. Proteins that putatively reduce Fd with hydrogen or pyruvate were enriched in anaerobes, while those that reduce Fd with NADH/NADPH were enriched in aerobes, facultative anaerobes, and anoxygenic phototrophs. The energy metabolism of aerobic, facultatively anaerobic, and anoxygenic phototrophic diazotrophs often yields reduced NADH/NADPH that is not sufficiently reduced to drive N2 reduction. At least two mechanisms have been acquired by these taxa to overcome this limitation and to generate electrons with potentials capable of reducing Fd. These include the bifurcation of electrons or the coupling of Fd reduction to reverse ion translocation.IMPORTANCE Nitrogen fixation supplies fixed nitrogen to cells from a variety of genomic and metabolic backgrounds including those of aerobes, facultative anaerobes, chemotrophs, and phototrophs. Here, using informatics approaches applied to genomic data, we show that pathways of electron transfer to nitrogenase in metabolically diverse diazotrophic taxa have diversified primarily in response to host cells' acquired ability to integrate O2 or light into their energy metabolism. Acquisition of two key enzyme complexes enabled aerobic and facultatively anaerobic phototrophic taxa to generate electrons of sufficiently low potential to reduce nitrogenase: the bifurcation of electrons via the Fix complex or the coupling of Fd reduction to reverse ion translocation via the Rhodobacter nitrogen fixation (Rnf) complex.
The path of electron transfer to nitrogenase in a phototrophic alpha-proteobacterium
(2018-07) Fixen, Kathryn R.; Pal Chowdhury, Nilanjan; Martinez-Perez, Marta; Poudel, Saroj; Boyd, Eric S.; Harwood, Caroline S.
The phototrophic alpha-proteobacterium, Rhodopseudomonas palustris, is a model for studies of regulatory and physiological parameters that control the activity of nitrogenase. This enzyme produces the energy-rich compound H2 , in addition to converting N2 gas to NH3 . Nitrogenase is an ATP-requiring enzyme that uses large amounts of reducing power, but the electron transfer pathway to nitrogenase in R. palustris was incompletely known. Here, we show that the ferredoxin, Fer1, is the primary but not sole electron carrier protein encoded by R. palustris that serves as an electron donor to nitrogenase. A flavodoxin, FldA, is also an important electron donor, especially under iron limitation. We present a model where the electron bifurcating complex, FixABCX, can reduce both ferredoxin and flavodoxin to transfer electrons to nitrogenase, and we present bioinformatic evidence that FixABCX and Fer1 form a conserved electron transfer pathway to nitrogenase in nitrogen-fixing proteobacteria. These results may be useful in the design of strategies to reroute electrons generated during metabolism of organic compounds to nitrogenase to achieve maximal activity.