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Direct electron-transfer anisotropy of a site-specifically immobilized cellobiose dehydrogenase

Direct electron-transfer anisotropy of a site-specifically immobilized cellobiose dehydrogenase
Direct electron-transfer anisotropy of a site-specifically immobilized cellobiose dehydrogenase
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.
Cellobiose dehydrogenase, direct electron transfer, site-specific mutagenesis, molecular modeling, anisotropic orientation, specific immobilization
2155-5435
7607-7615
Ma, Su
768041f5-4f85-4708-8604-698a5bd8430d
Laurent, Christophe V. F. P.
30620e53-30f7-412e-b4f7-2a69b9576190
Meneghello, Marta
0978ae7b-821d-49d9-9dac-a2a8e2306d5b
Tuoriniemi, Jani
dce5968c-ca35-46ce-9994-8b35af5b54fe
Oostenbrink, Chris
50785ff7-6f3c-4548-90a7-e57d210d537b
Gorton, Lo
2f9af82e-577d-495c-bbfe-a7775c7c583f
Bartlett, Philip N.
d99446db-a59d-4f89-96eb-f64b5d8bb075
Ludwig, Roland
9485312f-f31f-412f-85f0-cda0e34fb1aa
Ma, Su
768041f5-4f85-4708-8604-698a5bd8430d
Laurent, Christophe V. F. P.
30620e53-30f7-412e-b4f7-2a69b9576190
Meneghello, Marta
0978ae7b-821d-49d9-9dac-a2a8e2306d5b
Tuoriniemi, Jani
dce5968c-ca35-46ce-9994-8b35af5b54fe
Oostenbrink, Chris
50785ff7-6f3c-4548-90a7-e57d210d537b
Gorton, Lo
2f9af82e-577d-495c-bbfe-a7775c7c583f
Bartlett, Philip N.
d99446db-a59d-4f89-96eb-f64b5d8bb075
Ludwig, Roland
9485312f-f31f-412f-85f0-cda0e34fb1aa

Ma, Su, Laurent, Christophe V. F. P., Meneghello, Marta, Tuoriniemi, Jani, Oostenbrink, Chris, Gorton, Lo, Bartlett, Philip N. and Ludwig, Roland (2019) Direct electron-transfer anisotropy of a site-specifically immobilized cellobiose dehydrogenase. ACS Catalysis, 9 (8), 7607-7615. (doi:10.1021/acscatal.9b02014).

Record type: Article

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 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.

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Accepted/In Press date: 10 July 2019
e-pub ahead of print date: 10 July 2019
Keywords: Cellobiose dehydrogenase, direct electron transfer, site-specific mutagenesis, molecular modeling, anisotropic orientation, specific immobilization

Identifiers

Local EPrints ID: 433714
URI: http://eprints.soton.ac.uk/id/eprint/433714
ISSN: 2155-5435
PURE UUID: b3632cfd-7998-4738-902c-ab3a07ac1d1f
ORCID for Marta Meneghello: ORCID iD orcid.org/0000-0003-0099-1783
ORCID for Philip N. Bartlett: ORCID iD orcid.org/0000-0002-7300-6900

Catalogue record

Date deposited: 02 Sep 2019 16:30
Last modified: 26 Nov 2021 06:30

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Contributors

Author: Su Ma
Author: Christophe V. F. P. Laurent
Author: Marta Meneghello ORCID iD
Author: Jani Tuoriniemi
Author: Chris Oostenbrink
Author: Lo Gorton
Author: Roland Ludwig

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