Advanced

Direct Electron-Transfer Anisotropy of a Site-Specifically Immobilized Cellobiose Dehydrogenase

Ma, Su ; Laurent, Christophe V.F.P. ; Meneghello, Marta ; Tuoriniemi, Jani ; Oostenbrink, Chris ; Gorton, Lo LU ; Bartlett, Philip N. and Ludwig, Roland (2019) In ACS Catalysis p.7607-7615
Abstract

To study the direct electron transfer (DET) of the multicofactor enzyme cellobiose dehydrogenase (CDH) in regard to its orientation on an electrode surface, a recently published, maleimide-based immobilization method was used in combination with site-directed mutagenesis to establish different orientations on an electrode surface. CDH from Myriococcum thermophilum was chosen for this study because its protein structure is resolved and the factors influencing the movement of its mobile cytochrome domain (CYT) are established. Seven CDH variants with a surface-exposed cysteine residue in different spatial positions were generated for site-specific maleimide coupling. Surface plasmon resonance and cyclic voltammetry showed that all CDH... (More)

To study the direct electron transfer (DET) of the multicofactor enzyme cellobiose dehydrogenase (CDH) in regard to its orientation on an electrode surface, a recently published, maleimide-based immobilization method was used in combination with site-directed mutagenesis to establish different orientations on an electrode surface. CDH from Myriococcum thermophilum was chosen for this study because its protein structure is resolved and the factors influencing the movement of its mobile cytochrome domain (CYT) are established. Seven CDH variants with a surface-exposed cysteine residue in different spatial positions were generated for site-specific maleimide coupling. Surface plasmon resonance and cyclic voltammetry showed that all CDH variants, but not the wild-type CDH, bound covalently to gold electrodes or glassy carbon electrodes and were catalytically active. For DET, the CYT domain needs to move from the closed-state conformation, where it obtains an electron from the catalytic flavin adenine dinucleotide (FAD) cofactor to the open state where it can donate an electron to the electrode. We therefore hypothesized that the mobility of the CYT domain and its distance to the electrode is central for DET. We found that the uniform spatial orientations of CDH influenced DET as follows: an orientation of the two-domain enzyme on the side, with CYT in proximity to the electrode, resulted in high DET currents. Orientations with a bigger distance between CYT and the electrode, or orientations where CYT could not swing back to the dehydrogenase domain to form the closed enzyme conformation, reduced DET. In the latter case, calcium ions that stabilize the closed conformation of CDH fully recovered DET. The study demonstrates that a mobile CYT domain can compensate unfavorable orientations of the catalytic domain to a great extent and allows CDH as a multicofactor enzyme to transfer electrons even in awkward orientations. The mobile CYT domain reduces the anisotropy of DET, which is also essential for CDH's physiological function as an extracellular, electron-transferring enzyme.

(Less)
Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
anisotropic orientation, cellobiose dehydrogenase, direct electron transfer, molecular modeling, site-specific immobilization, site-specific mutagenesis
in
ACS Catalysis
pages
7607 - 7615
publisher
The American Chemical Society (ACS)
external identifiers
  • scopus:85070601938
ISSN
2155-5435
DOI
10.1021/acscatal.9b02014
language
English
LU publication?
yes
id
51a527fe-e02e-4a13-9f4b-fbcc39d62d80
date added to LUP
2019-08-26 15:03:28
date last changed
2019-09-17 04:59:24
@article{51a527fe-e02e-4a13-9f4b-fbcc39d62d80,
  abstract     = {<p>To study the direct electron transfer (DET) of the multicofactor enzyme cellobiose dehydrogenase (CDH) in regard to its orientation on an electrode surface, a recently published, maleimide-based immobilization method was used in combination with site-directed mutagenesis to establish different orientations on an electrode surface. CDH from Myriococcum thermophilum was chosen for this study because its protein structure is resolved and the factors influencing the movement of its mobile cytochrome domain (CYT) are established. Seven CDH variants with a surface-exposed cysteine residue in different spatial positions were generated for site-specific maleimide coupling. Surface plasmon resonance and cyclic voltammetry showed that all CDH variants, but not the wild-type CDH, bound covalently to gold electrodes or glassy carbon electrodes and were catalytically active. For DET, the CYT domain needs to move from the closed-state conformation, where it obtains an electron from the catalytic flavin adenine dinucleotide (FAD) cofactor to the open state where it can donate an electron to the electrode. We therefore hypothesized that the mobility of the CYT domain and its distance to the electrode is central for DET. We found that the uniform spatial orientations of CDH influenced DET as follows: an orientation of the two-domain enzyme on the side, with CYT in proximity to the electrode, resulted in high DET currents. Orientations with a bigger distance between CYT and the electrode, or orientations where CYT could not swing back to the dehydrogenase domain to form the closed enzyme conformation, reduced DET. In the latter case, calcium ions that stabilize the closed conformation of CDH fully recovered DET. The study demonstrates that a mobile CYT domain can compensate unfavorable orientations of the catalytic domain to a great extent and allows CDH as a multicofactor enzyme to transfer electrons even in awkward orientations. The mobile CYT domain reduces the anisotropy of DET, which is also essential for CDH's physiological function as an extracellular, electron-transferring enzyme.</p>},
  author       = {Ma, Su and Laurent, Christophe V.F.P. and Meneghello, Marta and Tuoriniemi, Jani and Oostenbrink, Chris and Gorton, Lo and Bartlett, Philip N. and Ludwig, Roland},
  issn         = {2155-5435},
  language     = {eng},
  pages        = {7607--7615},
  publisher    = {The American Chemical Society (ACS)},
  series       = {ACS Catalysis},
  title        = {Direct Electron-Transfer Anisotropy of a Site-Specifically Immobilized Cellobiose Dehydrogenase},
  url          = {http://dx.doi.org/10.1021/acscatal.9b02014},
  doi          = {10.1021/acscatal.9b02014},
  year         = {2019},
}