Comparative analysis of CO2 reduction by soluble Escherichia coli formate dehydrogenase H and its selenocysteine-to-cysteine substitution variant

Feilong Li; Silvan Scheller; Michael Lienemann

doi:10.1016/j.jcou.2023.102608

Comparative analysis of CO2 reduction by soluble Escherichia coli formate dehydrogenase H and its selenocysteine-to-cysteine substitution variant

Feilong Li
, Silvan Scheller
, Michael Lienemann^*

^*Corresponding author for this work

Aalto University

Research output: Contribution to journal › Article › Scientific › peer-review

9 Citations (Scopus)

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Abstract

Metal-dependent formate dehydrogenases (Me-FDHs) are highly active CO₂-reducing enzymes operating at low redox potentials and employ either molybdenum or tungsten to reduce the bound substrate. This makes them suitable for electrochemical applications such as fossil-free production of commodity chemicals utilizing renewable energy. Electrocatalytic CO₂ reduction by cathode-immobilized Me-FDHs has been recently demonstrated and rational protein engineering can be used to optimize Me-FDHs for various carbon reduction reactions. In the present study, CO₂ reduction by soluble monomeric Escherichia coli formate dehydrogenase H (EcFDH-H) was demonstrated and the function of its nucleophilic selenocysteine residue as a transient ligand of a centrally bound molybdenum atom was investigated. Kinetic analysis of the wildtype enzyme revealed maximum CO₂ reduction rates of 44 ± 6 s−1 at pH 5.8 that was decreased to 19% and 0% in the case of selenocysteine substitution with the structural homologues cysteine and serine, respectively. Further selenocysteine-to-cysteine substitution effects included an increased acid tolerance as well as stronger inhibition by nitrate and azide indicating a shift of the Mo oxidation state from IV to VI. Conversely, a destabilizing effect on the oxidized Mo(VI) center could be assigned to the native selenocysteine residue that may facilitate the observed efficient CO₂ reduction by rapid transition between Mo oxidation states. Taken together, the performed characterization of EcFDH-H as a catalyst for CO₂ reduction and the selenocysteine substitution analysis furthers the understanding of the active-site structure of Me-FDHs and thereby supports the development of more efficient biocatalysts for CO₂ reduction.

Original language	English
Article number	102608
Journal	Journal of CO2 Utilization
Volume	77
DOIs	https://doi.org/10.1016/j.jcou.2023.102608
Publication status	Published - Nov 2023
MoE publication type	A1 Journal article-refereed

Funding

We acknowledge financial support from the Research Council of Finland (formerly known as Academy of Finland) in the form of an Academy Research Fellowship grant awarded to M.L. and funding for the ExtremoForm project granted to S.S. (Decision Nos. 321723 and 329510 , respectively). The authors thank Prof. Shelley D. Minteer for the kind donation of the E. coli strain JG-X. Drs. William E. Robinson of Radboud University and Arnau Bassegoda of the Polytechnic University of Catalonia are thanked for their useful advice for EcFDH-H production. In addition, the assistance provided by Dr. Harry Boer of VTT Technical Research Centre of Finland in revising the manuscript is greatly appreciated. We acknowledge financial support from the Research Council of Finland (formerly known as Academy of Finland) in the form of an Academy Research Fellowship grant awarded to M.L. and funding for the ExtremoForm project granted to S.S. (Decision Nos. 321723 and 329510, respectively). The authors thank Prof. Shelley D. Minteer for the kind donation of the E. coli strain JG-X. Drs. William E. Robinson of Radboud University and Arnau Bassegoda of the Polytechnic University of Catalonia are thanked for their useful advice for EcFDH-H production. In addition, the assistance provided by Dr. Harry Boer of VTT Technical Research Centre of Finland in revising the manuscript is greatly appreciated.

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

SDG 7 Affordable and Clean Energy
SDG 13 Climate Action

Keywords

Carbon dioxide reduction
Selenocysteine
Formate dehydrogenase
Metalloenzyme
Molybdenum

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title = "Comparative analysis of CO2 reduction by soluble Escherichia coli formate dehydrogenase H and its selenocysteine-to-cysteine substitution variant",

abstract = "Metal-dependent formate dehydrogenases (Me-FDHs) are highly active CO2-reducing enzymes operating at low redox potentials and employ either molybdenum or tungsten to reduce the bound substrate. This makes them suitable for electrochemical applications such as fossil-free production of commodity chemicals utilizing renewable energy. Electrocatalytic CO2 reduction by cathode-immobilized Me-FDHs has been recently demonstrated and rational protein engineering can be used to optimize Me-FDHs for various carbon reduction reactions. In the present study, CO2 reduction by soluble monomeric Escherichia coli formate dehydrogenase H (EcFDH-H) was demonstrated and the function of its nucleophilic selenocysteine residue as a transient ligand of a centrally bound molybdenum atom was investigated. Kinetic analysis of the wildtype enzyme revealed maximum CO2 reduction rates of 44 ± 6 s−1 at pH 5.8 that was decreased to 19\% and 0\% in the case of selenocysteine substitution with the structural homologues cysteine and serine, respectively. Further selenocysteine-to-cysteine substitution effects included an increased acid tolerance as well as stronger inhibition by nitrate and azide indicating a shift of the Mo oxidation state from IV to VI. Conversely, a destabilizing effect on the oxidized Mo(VI) center could be assigned to the native selenocysteine residue that may facilitate the observed efficient CO2 reduction by rapid transition between Mo oxidation states. Taken together, the performed characterization of EcFDH-H as a catalyst for CO2 reduction and the selenocysteine substitution analysis furthers the understanding of the active-site structure of Me-FDHs and thereby supports the development of more efficient biocatalysts for CO2 reduction.",

keywords = "Carbon dioxide reduction, Selenocysteine, Formate dehydrogenase, Metalloenzyme, Molybdenum",

author = "Feilong Li and Silvan Scheller and Michael Lienemann",

year = "2023",

month = nov,

doi = "10.1016/j.jcou.2023.102608",

language = "English",

volume = "77",

journal = "Journal of CO2 Utilization",

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T1 - Comparative analysis of CO2 reduction by soluble Escherichia coli formate dehydrogenase H and its selenocysteine-to-cysteine substitution variant

AU - Li, Feilong

AU - Scheller, Silvan

AU - Lienemann, Michael

PY - 2023/11

Y1 - 2023/11

N2 - Metal-dependent formate dehydrogenases (Me-FDHs) are highly active CO2-reducing enzymes operating at low redox potentials and employ either molybdenum or tungsten to reduce the bound substrate. This makes them suitable for electrochemical applications such as fossil-free production of commodity chemicals utilizing renewable energy. Electrocatalytic CO2 reduction by cathode-immobilized Me-FDHs has been recently demonstrated and rational protein engineering can be used to optimize Me-FDHs for various carbon reduction reactions. In the present study, CO2 reduction by soluble monomeric Escherichia coli formate dehydrogenase H (EcFDH-H) was demonstrated and the function of its nucleophilic selenocysteine residue as a transient ligand of a centrally bound molybdenum atom was investigated. Kinetic analysis of the wildtype enzyme revealed maximum CO2 reduction rates of 44 ± 6 s−1 at pH 5.8 that was decreased to 19% and 0% in the case of selenocysteine substitution with the structural homologues cysteine and serine, respectively. Further selenocysteine-to-cysteine substitution effects included an increased acid tolerance as well as stronger inhibition by nitrate and azide indicating a shift of the Mo oxidation state from IV to VI. Conversely, a destabilizing effect on the oxidized Mo(VI) center could be assigned to the native selenocysteine residue that may facilitate the observed efficient CO2 reduction by rapid transition between Mo oxidation states. Taken together, the performed characterization of EcFDH-H as a catalyst for CO2 reduction and the selenocysteine substitution analysis furthers the understanding of the active-site structure of Me-FDHs and thereby supports the development of more efficient biocatalysts for CO2 reduction.

AB - Metal-dependent formate dehydrogenases (Me-FDHs) are highly active CO2-reducing enzymes operating at low redox potentials and employ either molybdenum or tungsten to reduce the bound substrate. This makes them suitable for electrochemical applications such as fossil-free production of commodity chemicals utilizing renewable energy. Electrocatalytic CO2 reduction by cathode-immobilized Me-FDHs has been recently demonstrated and rational protein engineering can be used to optimize Me-FDHs for various carbon reduction reactions. In the present study, CO2 reduction by soluble monomeric Escherichia coli formate dehydrogenase H (EcFDH-H) was demonstrated and the function of its nucleophilic selenocysteine residue as a transient ligand of a centrally bound molybdenum atom was investigated. Kinetic analysis of the wildtype enzyme revealed maximum CO2 reduction rates of 44 ± 6 s−1 at pH 5.8 that was decreased to 19% and 0% in the case of selenocysteine substitution with the structural homologues cysteine and serine, respectively. Further selenocysteine-to-cysteine substitution effects included an increased acid tolerance as well as stronger inhibition by nitrate and azide indicating a shift of the Mo oxidation state from IV to VI. Conversely, a destabilizing effect on the oxidized Mo(VI) center could be assigned to the native selenocysteine residue that may facilitate the observed efficient CO2 reduction by rapid transition between Mo oxidation states. Taken together, the performed characterization of EcFDH-H as a catalyst for CO2 reduction and the selenocysteine substitution analysis furthers the understanding of the active-site structure of Me-FDHs and thereby supports the development of more efficient biocatalysts for CO2 reduction.

KW - Carbon dioxide reduction

KW - Selenocysteine

KW - Formate dehydrogenase

KW - Metalloenzyme

KW - Molybdenum

UR - https://www.scopus.com/pages/publications/85174333009

U2 - 10.1016/j.jcou.2023.102608

DO - 10.1016/j.jcou.2023.102608

M3 - Article

SN - 2212-9820

VL - 77

JO - Journal of CO2 Utilization

JF - Journal of CO2 Utilization

M1 - 102608

ER -

Comparative analysis of CO2 reduction by soluble Escherichia coli formate dehydrogenase H and its selenocysteine-to-cysteine substitution variant

Abstract

Funding

UN SDGs

Keywords

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