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The influence of pH and divalent/monovalent cations on the internal electron transfer (IET), enzymatic activity, and structure of fructose dehydrogenase

Bollella, Paolo; Hibino, Yuya; Kano, Kenji; Gorton, Lo LU and Antiochia, Riccarda (2018) In Analytical and Bioanalytical Chemistry p.1-12
Abstract

We report on the influence of pH and monovalent/divalent cations on the catalytic current response, internal electron transfer (IET), and structure of fructose dehydrogenase (FDH) by using amperometry, spectrophotometry, and circular dichroism (CD). Amperometric measurements were performed on graphite electrodes, onto which FDH was adsorbed and the effect on the response current to fructose was investigated when varying the pH and the concentrations of divalent/monovalent cations in the contacting buffer. In the presence of 10 mM CaCl2, a current increase of up to ≈ 240% was observed, probably due to an intra-complexation reaction between Ca2+ and the aspartate/glutamate residues found at the interface between the... (More)

We report on the influence of pH and monovalent/divalent cations on the catalytic current response, internal electron transfer (IET), and structure of fructose dehydrogenase (FDH) by using amperometry, spectrophotometry, and circular dichroism (CD). Amperometric measurements were performed on graphite electrodes, onto which FDH was adsorbed and the effect on the response current to fructose was investigated when varying the pH and the concentrations of divalent/monovalent cations in the contacting buffer. In the presence of 10 mM CaCl2, a current increase of up to ≈ 240% was observed, probably due to an intra-complexation reaction between Ca2+ and the aspartate/glutamate residues found at the interface between the dehydrogenase domain and the cytochrome domain of FDH. Contrary to CaCl2, addition of MgCl2 did not show any particular influence, whereas addition of monovalent cations (Na+ or K+) led to a slight linear increase in the maximum response current. To complement the amperometric investigations, spectrophotometric assays were carried out under homogeneous conditions in the presence of a 1-electron non-proton-acceptor, cytochrome c, or a 2-electron-proton acceptor, 2,6-dichloroindophenol (DCIP), respectively. In the case of cytochrome c, it was possible to observe a remarkable increase in the absorbance up to 200% when 10 mM CaCl2 was added. However, by further increasing the concentration of CaCl2 up to 50 mM and 100 mM, a decrease in the absorbance with a slight inhibition effect was observed for the highest CaCl2 concentration. Addition of MgCl2 or of the monovalent cations shows, surprisingly, no effect on the electron transfer to the electron acceptor. Contrary to the case of cytochrome c, with DCIP none of the cations tested seem to affect the rate of catalysis. In order to correlate the results obtained by amperometric and spectrophotometric measurements, CD experiments have been performed showing a great structural change of FDH when increasing the concentration CaCl2 up to 50 mM, at which the enzyme molecules start to agglomerate, hindering the substrate access to the active site probably due to a chelation reaction occurring at the enzyme surface with the glutamate/aspartate residues. [Figure not available: see fulltext.]

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author
organization
publishing date
type
Contribution to journal
publication status
epub
subject
keywords
Calcium chloride, Direct electron transfer (DET), Enzyme activity, Enzyme structure, Fructose dehydrogenase (FDH)
in
Analytical and Bioanalytical Chemistry
pages
12 pages
publisher
Springer
external identifiers
  • scopus:85044210561
ISSN
1618-2642
DOI
10.1007/s00216-018-0991-0
language
English
LU publication?
yes
id
6400741d-f22e-44d5-9e13-5c7d678dc56e
date added to LUP
2018-04-03 14:38:44
date last changed
2018-07-08 04:30:54
@article{6400741d-f22e-44d5-9e13-5c7d678dc56e,
  abstract     = {<p>We report on the influence of pH and monovalent/divalent cations on the catalytic current response, internal electron transfer (IET), and structure of fructose dehydrogenase (FDH) by using amperometry, spectrophotometry, and circular dichroism (CD). Amperometric measurements were performed on graphite electrodes, onto which FDH was adsorbed and the effect on the response current to fructose was investigated when varying the pH and the concentrations of divalent/monovalent cations in the contacting buffer. In the presence of 10 mM CaCl<sub>2</sub>, a current increase of up to ≈ 240% was observed, probably due to an intra-complexation reaction between Ca<sup>2+</sup> and the aspartate/glutamate residues found at the interface between the dehydrogenase domain and the cytochrome domain of FDH. Contrary to CaCl<sub>2</sub>, addition of MgCl<sub>2</sub> did not show any particular influence, whereas addition of monovalent cations (Na<sup>+</sup> or K<sup>+</sup>) led to a slight linear increase in the maximum response current. To complement the amperometric investigations, spectrophotometric assays were carried out under homogeneous conditions in the presence of a 1-electron non-proton-acceptor, cytochrome c, or a 2-electron-proton acceptor, 2,6-dichloroindophenol (DCIP), respectively. In the case of cytochrome c, it was possible to observe a remarkable increase in the absorbance up to 200% when 10 mM CaCl<sub>2</sub> was added. However, by further increasing the concentration of CaCl<sub>2</sub> up to 50 mM and 100 mM, a decrease in the absorbance with a slight inhibition effect was observed for the highest CaCl<sub>2</sub> concentration. Addition of MgCl<sub>2</sub> or of the monovalent cations shows, surprisingly, no effect on the electron transfer to the electron acceptor. Contrary to the case of cytochrome c, with DCIP none of the cations tested seem to affect the rate of catalysis. In order to correlate the results obtained by amperometric and spectrophotometric measurements, CD experiments have been performed showing a great structural change of FDH when increasing the concentration CaCl<sub>2</sub> up to 50 mM, at which the enzyme molecules start to agglomerate, hindering the substrate access to the active site probably due to a chelation reaction occurring at the enzyme surface with the glutamate/aspartate residues. [Figure not available: see fulltext.]</p>},
  author       = {Bollella, Paolo and Hibino, Yuya and Kano, Kenji and Gorton, Lo and Antiochia, Riccarda},
  issn         = {1618-2642},
  keyword      = {Calcium chloride,Direct electron transfer (DET),Enzyme activity,Enzyme structure,Fructose dehydrogenase (FDH)},
  language     = {eng},
  month        = {03},
  pages        = {1--12},
  publisher    = {Springer},
  series       = {Analytical and Bioanalytical Chemistry},
  title        = {The influence of pH and divalent/monovalent cations on the internal electron transfer (IET), enzymatic activity, and structure of fructose dehydrogenase},
  url          = {http://dx.doi.org/10.1007/s00216-018-0991-0},
  year         = {2018},
}