Substrate–Protein Interactions of Type II NADH:Quinone Oxidoreductase from <i>Escherichia coli</i>

Salewski, Johannes; Batista, Ana P.; Sena, Filipa V.; Millo, Diego; Zebger, Ingo; Pereira, Manuela M.; Hildebrandt, Peter

doi:10.1021/acs.biochem.6b00070

Cited by 13 publications

(14 citation statements)

References 46 publications

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“…Here, the absorption of both NAD + (which has an absorption maximum at 260 nm that extends to 280 nm) and NADH (maximum absorption at 340 nm) precluded identification of any changes due to nucleotide binding. Significant fluorescence changes were not observed from the flavin in either S. aureus, C. thermarum, or Escherichia coli NDH-222, monitored at 450 and 530 nm (at which wavelengths the nucleotides do not absorb).…”

Section: Resultsmentioning

confidence: 95%

The mechanism of catalysis by type-II NADH:quinone oxidoreductases

Blaza

Bridges

Aragão

et al. 2017

Sci Rep

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Type II NADH:quinone oxidoreductase (NDH-2) is central to the respiratory chains of many organisms. It is not present in mammals so may be exploited as an antimicrobial drug target or used as a substitute for dysfunctional respiratory complex I in neuromuscular disorders. NDH-2 is a single-subunit monotopic membrane protein with just a flavin cofactor, yet no consensus exists on its mechanism. Here, we use steady-state and pre-steady-state kinetics combined with mutagenesis and structural studies to determine the mechanism of NDH-2 from Caldalkalibacillus thermarum. We show that the two substrate reactions occur independently, at different sites, and regardless of the occupancy of the partner site. We conclude that the reaction pathway is determined stochastically, by the substrate/product concentrations and dissociation constants, and can follow either a ping-pong or ternary mechanism. This mechanistic versatility provides a unified explanation for all extant data and a new foundation for the development of therapeutic strategies.

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Section: Resultsmentioning

confidence: 95%

The mechanism of catalysis by type-II NADH:quinone oxidoreductases

Blaza

Bridges

Aragão

et al. 2017

Sci Rep

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“…The interfacial electric field has also been pointed out to affect the redox potential of enzymes. A SEIRA study of the type II NADH: Quinone Oxidoreductase from E. coli immobilized on SAM-modified gold electrodes outlined a substantial increase in the redox potential of the enzyme (~140 mV) [191]. The authors attributed this upshift in potential to an electric-field-driven stabilization of the flavin cofactor.…”

Section: Effect Of Potential Electric Fieldmentioning

confidence: 99%

Controlling Redox Enzyme Orientation at Planar Electrodes

et al. 2018

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Abstract:Redox enzymes, which catalyze reactions involving electron transfers in living organisms, are very promising components of biotechnological devices, and can be envisioned for sensing applications as well as for energy conversion. In this context, one of the most significant challenges is to achieve efficient direct electron transfer by tunneling between enzymes and conductive surfaces. Based on various examples of bioelectrochemical studies described in the recent literature, this review discusses the issue of enzyme immobilization at planar electrode interfaces. The fundamental importance of controlling enzyme orientation, how to obtain such orientation, and how it can be verified experimentally or by modeling are the three main directions explored. Since redox enzymes are sizable proteins with anisotropic properties, achieving their functional immobilization requires a specific and controlled orientation on the electrode surface. All the factors influenced by this orientation are described, ranging from electronic conductivity to efficiency of substrate supply. The specificities of the enzymatic molecule, surface properties, and dipole moment, which in turn influence the orientation, are introduced. Various ways of ensuring functional immobilization through tuning of both the enzyme and the electrode surface are then described. Finally, the review deals with analytical techniques that have enabled characterization and quantification of successful achievement of the desired orientation. The rich contributions of electrochemistry, spectroscopy (especially infrared spectroscopy), modeling, and microscopy are featured, along with their limitations.

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“…The catalytic mechanism of NDH-2 is still unclear, even considering the available structural and functional data 4 5 6 7 8 12 . Nevertheless the gathered information showed that the two substrates bind to different sites, and that a charge-transfer complex is formed between NAD + and the reduced flavin (FADH 2 ), which is dissociated by the quinone 7 8 12 . Here, we discuss the possible roles of the conserved elements in the catalytic process ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Sena et al detected, for NDH-2 from S. aureus, a charge-transfer complex formed between NAD + and the reduced flavin, which is dissociated by the quinone 7 . Recently NDH-2 from Escherichia coli was also shown to have two distinct substrate binding sites and a bound semi-protonated quinol was identified as a catalytic intermediate 8 . All these recent findings were major advances for the understanding of NDH-2, but its overall mechanism is still unclear.…”

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confidence: 99%

Structural and Functional insights into the catalytic mechanism of the Type II NADH:quinone oxidoreductase family

Marreiros

Sena

Sousa

et al. 2017

Sci Rep

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Type II NADH:quinone oxidoreductases (NDH-2s) are membrane proteins involved in respiratory chains. These proteins contribute indirectly to the establishment of the transmembrane difference of electrochemical potential by catalyzing the reduction of quinone by oxidation of NAD(P)H. NDH-2s are widespread enzymes being present in the three domains of life. In this work, we explored the catalytic mechanism of NDH-2 by investigating the common elements of all NDH-2s, based on the rationale that conservation of such elements reflects their structural/functional importance. We observed conserved sequence motifs and structural elements among 1762 NDH-2s. We identified two proton pathways possibly involved in the protonation of the quinone. Our results led us to propose the first catalytic mechanism for NDH-2 family, in which a conserved glutamate residue, E172 (in NDH-2 from Staphylococcus aureus) plays a key role in proton transfer to the quinone pocket. This catalytic mechanism may also be extended to the other members of the two-Dinucleotide Binding Domains Flavoprotein (tDBDF) superfamily, such as sulfide:quinone oxidoreductases.

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Substrate–Protein Interactions of Type II NADH:Quinone Oxidoreductase from Escherichia coli

Cited by 13 publications

References 46 publications

The mechanism of catalysis by type-II NADH:quinone oxidoreductases

The mechanism of catalysis by type-II NADH:quinone oxidoreductases

Controlling Redox Enzyme Orientation at Planar Electrodes

Structural and Functional insights into the catalytic mechanism of the Type II NADH:quinone oxidoreductase family

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