Browsing by Author "Nguyen, Diep M. N."

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The catalytic mechanism of electron bifurcating electron transfer flavoproteins (ETFs) involves an intermediary complex with NAD
(2018-12) Schut, Gerrit J.; Mohamed-Raseek, Nishya; Tokmina-Lukaszewska, Monika; Mulder, David W.; Nguyen, Diep M. N.; Lipscomb, Gina L.; Hoben, John P.; Patterson, Angela; Lubner, Carolyn E.; King, Paul W.; Peters, John W.; Bothner, Brian; Miller, Anne-Frances; Adams, Michael W. W.
Electron bifurcation plays a key role in anaerobic energy metabolism but it is a relatively new discovery and only limited mechanistic information is available on the diverse enzymes that employ it. Herein, we focused on the bifurcating electron transfer flavoprotein (ETF) from the hyperthermophilic archaeon Pyrobaculum aerophilum The EtfABCX enzyme complex couples NADH oxidation to the endergonic reduction of ferredoxin and exergonic reduction of menaquinone. We developed a model for the enzyme structure by using non-denaturing MS, cross-linking and homology modeling in which EtfA, B, and C each contained FAD, whereas EtfX contained two [4Fe-4S] clusters. On the basis of analyses using transient absorption, EPR and optical titrations with NADH or inorganic reductants with and without NAD+, we propose a catalytic cycle involving formation of an intermediary NAD+-bound complex. A charge transfer signal revealed an intriguing interplay of flavin semiquinones and a protein conformational change that gated electron transfer between the low- and high-potential pathways. We found that despite a common bifurcating flavin site, the proposed EtfABCX catalytic cycle is distinct from that of the genetically-unrelated bifurcating NADH-dependent ferredoxin NADP+ oxidoreductase (NfnI). The two enzymes particularly differed in the role of NAD+, the resting and bifurcating-ready states of the enzymes, how electron flow is gated, and in the two two-electron cycles constituting the overall four-electron reaction. We conclude that P. aerophilum EtfABCX provides a model catalytic mechanism that builds on and extends previous studies of related bifurcating ETF\'s and can be applied to the large bifurcating ETF family.
H/D exchange mass spectrometry and statistical coupling analysis reveal a role for allostery in a ferredoxin-dependent bifurcating transhydrogenase catalytic cycle
(2018-01) Berry, Luke; Poudel, Saroj; Tokmina-Lukaszewska, Monika; Colman, Daniel R.; Nguyen, Diep M. N.; Schut, Gerrit J.; Adams, Michael W. W.; Peters, John W.; Boyd, Eric S.; Bothner, Brian
Recent investigations into ferredoxin-dependent transhydrogenases, a class of enzymes responsible for electron transport, have highlighted the biological importance of flavin-based electron bifurcation (FBEB). FBEB generates biomolecules with very low reduction potential by coupling the oxidation of an electron donor with intermediate potential to the reduction of high and low potential molecules. Bifurcating systems can generate biomolecules with very low reduction potentials, such as reduced ferredoxin (Fd), from species such as NADPH. Metabolic systems that use bifurcation are more efficient and confer a competitive advantage for the organisms that harbor them. Structural models are now available for two NADH-dependent ferredoxin-NADP(+) oxidoreductase (Nfn) complexes. These models, together with spectroscopic studies, have provided considerable insight into the catalytic process of FBEB. However, much about the mechanism and regulation of these multi-subunit proteins remains unclear. Using hydrogen/deuterium exchange mass spectrometry (HDX-MS) and statistical coupling analysis (SCA), we identified specific pathways of communication within the model FBEB system, Nfn from Pyrococus furiosus, under conditions at each step of the catalytic cycle. HDX-MS revealed evidence for allosteric coupling across protein subunits upon nucleotide and ferredoxin binding. SCA uncovered a network of co-evolving residues that can provide connectivity across the complex. Together, the HDX-MS and SCA data show that protein allostery occurs across the ensemble of iron-sulfur cofactors and ligand binding sites using specific pathways that connect domains allowing them to function as dynamically coordinated units.
Two functionally distinct NADP(+)-dependent ferredoxin oxidoreductases maintain the primary redox balance of Pyrococcus furiosus
(2017-07) Nguyen, Diep M. N.; Schut, Gerrit J.; Zadvornyy, Oleg A.; Tokmina-Lukaszewska, Monika; Poudel, Saroj; Lipscomb, Gina L.; Adams, Leslie A.; Dinsmore, Jessica T.; Nixon, William J.; Boyd, Eric S.; Bothner, Brian; Peters, John W.; Adams, Michael W. W.
Electron bifurcation has recently gained acceptance as the third mechanism of energy conservation in which energy is conserved through the coupling of exergonic and endergonic reactions. A structure-based mechanism of bifurcation has been elucidated recently for the flavin-based enzyme NADH-dependent ferredoxin NADP+ oxidoreductase I (NfnI) from the hyperthermophillic archaeon Pyrococcus furiosus. NfnI is thought to be involved in maintaining the cellular redox balance, producing NADPH for biosynthesis by recycling the two other primary redox carriers, NADH and ferredoxin. The P. furiosus genome encodes an NfnI paralog termed NfnII, and the two are differentially expressed depending on the growth conditions. In this study, we show that deletion of the genes encoding either NfnI or NfnII affects the cellular concentrations of NAD(P)H and particularly NADPH. This results in a moderate to severe growth phenotype in deletion mutants, demonstrating a key role for each enzyme in maintaining redox homeostasis. Despite their similarity in primary sequence and cofactor content, crystallographic, kinetic, and mass spectrometry analyses reveal that there are fundamental structural differences between the two enzymes and NfnII does not catalyze the NfnI bifurcating reaction. Instead it exhibits non-bifurcating ferredoxin NADP oxidoreductase-type activity. NfnII is therefore proposed to be a bifunctional enzyme and to also catalyze a bifurcating reaction, although its third substrate, in addition to ferredoxin and NADP(H), is as yet unknown.