Insights into Flavin-based Electron Bifurcation via the NADH-dependent Reduced Ferredoxin:NADP Oxidoreductase Structure

Demmer, Julius K.; Huang, Haiyan; Wang, Shuning; Demmer, Ulrike; Thauer, Rudolf K.; Ermler, Ulrich

doi:10.1074/jbc.m115.656520

Cited by 95 publications

(134 citation statements)

References 52 publications

(54 reference statements)

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“…As demonstrated biochemically, the NfnB protein is part of a heterodimeric complex that accepts electrons from NADH and ferredoxin during electron bifurcation that produces NADPH (Demmer et al, 2015). Since our mechanism is essentially the reverse of the NfnAB reaction, sequence homology provides circumstantial support for an analogous function.…”

Section: (Nz_cp011308)mentioning

confidence: 93%

Productivity, metabolism and physiology of free-living chemoautotrophic Epsilonproteobacteria

McNichol¹

2016

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Chemoautotrophic ecosystems at deep-sea hydrothermal vents were discovered in 1977, but not until 1995 were free-living autotrophic Epsilonproteobacteria identified as important microbial community members. Because the deep-sea is food-starved, the autotrophic metabolism of hydrothermal vent Epsilonproteobacteria may be very important for deep-sea consumers. However, quantifying their metabolic activities in situ has remained difficult, and biochemical mechanisms underlying their autotrophic physiology are poorly described. To gain insight into environmental processes, an approach was developed for incubations of microbes at in situ pressure and temperature (25 MPa, 24 o C) with various combinations of electron donors/acceptors (H 2 , O 2 and NO 3 -and 13 HCO 3 -) as a tracer to track carbon fixation. During short (18-24 h) incubations of low-temperature vent fluids from Crab Spa (9 o N East Pacific Rise), the concentration of electron donors/acceptors and cell numbers were monitored to quantify microbial processes. Measured rates were generally higher than previous studies, and the stoichiometry of microbially-catalyzed redox reactions revealed new insights into sulfur and nitrogen cycling. Single-cell, taxonomically-resolved tracer incorporation showed Epsilonproteobacteria dominated carbon fixation, and their growth efficiency was calculated based on electron acceptor consumption. Using these data, in situ primary productivity, microbial standing stock, and average biomass residence time of the deep-sea vent subseafloor biosphere were estimated. Finally, the population structures of the most abundant genera Sulfurimonas and Thioreductor were shown to be strongly influenced by pO 2 and temperature respectively, providing a mechanism for niche differentiation in situ. To gain insights into the core biochemical reactions underlying autotrophy in Epsilonprotebacteria, a theoretical metabolic model of Sulfurimonas denitrificans was developed. Validated iteratively by comparing in silico yields with data from chemostat experiments, the model generated hypotheses explaining critical, yet so far unresolved reactions supporting chemoautotrophy in Epsilonproteobacteria. For example, it provides insight into how energy is conserved during sulfur oxidation coupled to denitrification, how reverse electron transport produces ferredoxin for carbon fixation, and why aerobic growth yields are only slightly higher compared to denitrification. As a whole, this thesis provides important contributions towards understanding core mechanisms of chemoautrophy, as well as the in situ productivity, physiology and ecology of autotrophic Epsilonproteobacteria.

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Section: (Nz_cp011308)mentioning

confidence: 93%

Productivity, metabolism and physiology of free-living chemoautotrophic Epsilonproteobacteria

McNichol¹

2016

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“…This suggests that regardless of the direction in which they operate, we can expect bifurcating enzymes to possess at least three redox cofactors, one of which will serve as the transducer between 2-electron and 1-electron reactions at 150 Bioinorganic Chemistry Hypothetical mechanism of electron transfer in NADH-dependent reduced ferredoxin:NADP + oxidoreductase (Nfn). This model is adapted from [16 ]. Electrons added to Nfn are indicated and counted in green, those leaving are in red.…”

Section: Flavins In Bifurcationmentioning

confidence: 99%

Electron bifurcation

Peters

Miller

Jones

et al. 2016

Current Opinion in Chemical Biology

125

121

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Electron bifurcation is the recently recognized third mechanism of biological energy conservation. It simultaneously couples exergonic and endergonic oxidation-reduction reactions to circumvent thermodynamic barriers and minimize free energy loss. Little is known about the details of how electron bifurcating enzymes function, but specifics are beginning to emerge for several bifurcating enzymes. To date, those characterized contain a collection of redox cofactors including flavins and iron-sulfur clusters. Here we discuss the current understanding of bifurcating enzymes and the mechanistic features required to reversibly partition multiple electrons from a single redox site into exergonic and endergonic electron transfer paths.

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“…For this assay, the apparent K m for NAD ϩ was found to be approximately 0.5 mM and the apparent V max was found to be 3.28 U per mg of protein at 55°C and pH 7.5. Tsac_1705 protein was annotated as a dihydroorotate dehydrogenase (DodH) electron transfer subunit and is part of a putative operon for dihydroorotate dehydrogenase, it is similar (32% amino acid sequence identity) to the NfnA protein, one of the subunits of the NfnAB complex from Thermotoga maritima with known NFN activity (20). The most similar protein with an available crystal structure is DodH from Lactococcus lactis, with a sequence identity of 39% (35 (Fig.…”

Section: Identification Of Ferredoxin:nadmentioning

confidence: 99%

“…Another coupling reaction is the transhydrogenation reaction, resulting in the NADH-dependent reduced FNOR reaction (Fig. 1, equation c) (10,19,20).…”

mentioning

confidence: 99%

“…Another coupling reaction is the transhydrogenation reaction, resulting in the NADH-dependent reduced FNOR reaction (Fig. 1, equation c) (10,19,20).Thermoanaerobacterium saccharolyticum is a thermophilic bacterium with the ability to ferment many components of the hemicellulose fraction of lignocellulosic biomass, including xylan, and can produce ethanol at high yields and titers. Engineered strains of T. saccharolyticum that can produce over 70 g/liter ethanol at neartheoretical yield have been developed (21), and this has inspired us to study the pathway this organism uses to produce ethanol.…”

mentioning

confidence: 99%

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Ferredoxin:NAD ⁺ Oxidoreductase of Thermoanaerobacterium saccharolyticum and Its Role in Ethanol Formation

Tian

Shao

et al. 2016

Appl Environ Microbiol

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Ferredoxin:NAD؉ oxidoreductase (NADH-FNOR) catalyzes the transfer of electrons from reduced ferredoxin to NAD ؉ . This enzyme has been hypothesized to be the main enzyme responsible for ferredoxin oxidization in the NADH-based ethanol pathway in Thermoanaerobacterium saccharolyticum; however, the corresponding gene has not yet been identified. Here, we identified the Tsac_1705 protein as a candidate FNOR based on the homology of its functional domains. We then confirmed its activity in vitro with a ferredoxin-based FNOR assay. To determine its role in metabolism, the tsac_1705 gene was deleted in different strains of T. saccharolyticum. In wild-type T. saccharolyticum, deletion of tsac_1705 resulted in a 75% loss of NADH-FNOR activity, which indicated that Tsac_1705 is the main NADH-FNOR in T. saccharolyticum. When both NADH-and NADPH-linked FNOR genes were deleted, the ethanol titer decreased and the ratio of ethanol to acetate approached unity, indicative of the absence of FNOR activity. Finally, we tested the effect of heterologous expression of Tsac_1705 in Clostridium thermocellum and found improvements in both the titer and the yield of ethanol. IMPORTANCERedox balance plays a crucial role in many metabolic engineering strategies. Ferredoxins are widely used as electron carriers for anaerobic microorganism and plants. This study identified the gene responsible for electron transfer from ferredoxin to NAD ؉ , a key reaction in the ethanol production pathway of this organism and many other metabolic pathways. Identification of this gene is an important step in transferring the ethanol production ability of this organism to other organisms. Ferredoxins are iron-sulfur proteins found in many anaerobic bacteria and archaea and mediate electron transfer in various metabolic processes, including photosynthesis (1, 2), alcohol production (3, 4), nitrogen fixation (5, 6), and hydrogen production (7). Lack of knowledge about these ferredoxin-dependent pathways currently limits our ability to incorporate them into metabolic engineering strategies. The ferredoxin:NAD ϩ /NADP ϩ oxidoreductase (FNOR) enzyme (EC 1.18.1.2/EC 1.18.1.3) forms a key bridge in metabolism between the nicotinamide cofactor (i.e., NAD ϩ , NADH, NADP ϩ , and NADPH)-dependent pathways and ferredoxin-dependent pathways (Fig. 1, equation a) (8-11). Recently, the thioredoxin reductase-like (TrxR-type) FNORs were found, and they are widely distributed among the bacteria and archaea (12)(13)(14). Even these types of FNORs are more homologous to bacterial NADPH-TrxRs, but they have catalytic properties similar to those of FNOR (14). FNOR enzymes are widely believed to be of central importance for the bioenergetics of anaerobic bacteria due to their ability to couple electron transport with ion or Na ϩ gradient generation (15) or transhydrogenation. Furthermore, they are the key enzymes for many biochemical and biofuel pathways, including isopropanol, ethanol, and n-butanol (3,4,16). Since ferredoxin has a lower standard reduction potential than...

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Insights into Flavin-based Electron Bifurcation via the NADH-dependent Reduced Ferredoxin:NADP Oxidoreductase Structure

Cited by 95 publications

References 52 publications

Productivity, metabolism and physiology of free-living chemoautotrophic Epsilonproteobacteria

Productivity, metabolism and physiology of free-living chemoautotrophic Epsilonproteobacteria

Electron bifurcation

Ferredoxin:NAD ⁺ Oxidoreductase of Thermoanaerobacterium saccharolyticum and Its Role in Ethanol Formation

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Insights into Flavin-based Electron Bifurcation via the NADH-dependent Reduced Ferredoxin:NADP Oxidoreductase Structure

Cited by 95 publications

References 52 publications

Productivity, metabolism and physiology of free-living chemoautotrophic Epsilonproteobacteria

Productivity, metabolism and physiology of free-living chemoautotrophic Epsilonproteobacteria

Electron bifurcation

Ferredoxin:NAD + Oxidoreductase of Thermoanaerobacterium saccharolyticum and Its Role in Ethanol Formation

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Ferredoxin:NAD ⁺ Oxidoreductase of Thermoanaerobacterium saccharolyticum and Its Role in Ethanol Formation