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Title: Effect of a C298D Mutation in CaHydA [FeFe]-Hydrogenase: Insights into the Protein-Metal Cluster Interaction by EPR and FTIR Spectroscopic Investigation

Abstract

A conserved cysteine located in the signature motif of the catalytic center (H-cluster) of [FeFe]-hydrogenases functions in proton transfer. This residue corresponds to C298 in Clostridium acetobutylicum CaHydA. Despite the chemical and structural difference, the mutant C298D retains fast catalytic activity, while replacement with any other amino acid caused significant activity loss. Given the proximity of C298 to the H-cluster, the effect of the C298D mutation on the catalytic center was studied by continuous wave (CW) and pulse electron paramagnetic resonance (EPR) and by Fourier transform infrared (FTIR) spectroscopies. Comparison of the C298D mutant with the wild type CaHydA by CW and pulse EPR showed that the electronic structure of the center is not altered. FTIR spectroscopy confirmed that absorption peak values observed in the mutant are virtually identical to those observed in the wild type, indicating that the H-cluster is not generally affected by the mutation. Significant differences were observed only in the inhibited state Hox-CO: the vibrational modes assigned to the COexo and Fed-CO in this state are shifted to lower values in C298D, suggesting different interaction of these ligands with the protein moiety when C298 is changed to D298. More relevant to the catalytic cycle, the redoxmore » equilibrium between the Hox and Hred states is modified by the mutation, causing a prevalence of the oxidized state. This work highlights how the interactions between the protein environment and the H-cluster, a dynamic closely interconnected system, can be engineered and studied in the perspective of designing bio-inspired catalysts and mimics.« less

Authors:
; ; ; ; ; ; ; ;
Publication Date:
Research Org.:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES)
OSTI Identifier:
1234222
Report Number(s):
NREL/JA-2700-65336
Journal ID: ISSN 0005-2728
DOE Contract Number:  
AC36-08GO28308
Resource Type:
Journal Article
Journal Name:
Biochimica et Biophysica Acta - Bioenergetics
Additional Journal Information:
Journal Volume: 1857; Journal Issue: 1; Related Information: Biochimica et Biophysica Acta (BBA) - Bioenergetics; Journal ID: ISSN 0005-2728
Publisher:
Elsevier
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANAYLYTICAL CHEMISTRY; [FeFe]-hydrogenase; proton transfer; EPR; HYSCORE; FTIR

Citation Formats

Morra, Simone, Maurelli, Sara, Chiesa, Mario, Mulder, David W., Ratzloff, Michael W., Giamello, Elio, King, Paul W., Gilardi, Gianfranco, and Valettia, Francesca. Effect of a C298D Mutation in CaHydA [FeFe]-Hydrogenase: Insights into the Protein-Metal Cluster Interaction by EPR and FTIR Spectroscopic Investigation. United States: N. p., 2016. Web. doi:10.1016/j.bbabio.2015.10.005.
Morra, Simone, Maurelli, Sara, Chiesa, Mario, Mulder, David W., Ratzloff, Michael W., Giamello, Elio, King, Paul W., Gilardi, Gianfranco, & Valettia, Francesca. Effect of a C298D Mutation in CaHydA [FeFe]-Hydrogenase: Insights into the Protein-Metal Cluster Interaction by EPR and FTIR Spectroscopic Investigation. United States. https://doi.org/10.1016/j.bbabio.2015.10.005
Morra, Simone, Maurelli, Sara, Chiesa, Mario, Mulder, David W., Ratzloff, Michael W., Giamello, Elio, King, Paul W., Gilardi, Gianfranco, and Valettia, Francesca. 2016. "Effect of a C298D Mutation in CaHydA [FeFe]-Hydrogenase: Insights into the Protein-Metal Cluster Interaction by EPR and FTIR Spectroscopic Investigation". United States. https://doi.org/10.1016/j.bbabio.2015.10.005.
@article{osti_1234222,
title = {Effect of a C298D Mutation in CaHydA [FeFe]-Hydrogenase: Insights into the Protein-Metal Cluster Interaction by EPR and FTIR Spectroscopic Investigation},
author = {Morra, Simone and Maurelli, Sara and Chiesa, Mario and Mulder, David W. and Ratzloff, Michael W. and Giamello, Elio and King, Paul W. and Gilardi, Gianfranco and Valettia, Francesca},
abstractNote = {A conserved cysteine located in the signature motif of the catalytic center (H-cluster) of [FeFe]-hydrogenases functions in proton transfer. This residue corresponds to C298 in Clostridium acetobutylicum CaHydA. Despite the chemical and structural difference, the mutant C298D retains fast catalytic activity, while replacement with any other amino acid caused significant activity loss. Given the proximity of C298 to the H-cluster, the effect of the C298D mutation on the catalytic center was studied by continuous wave (CW) and pulse electron paramagnetic resonance (EPR) and by Fourier transform infrared (FTIR) spectroscopies. Comparison of the C298D mutant with the wild type CaHydA by CW and pulse EPR showed that the electronic structure of the center is not altered. FTIR spectroscopy confirmed that absorption peak values observed in the mutant are virtually identical to those observed in the wild type, indicating that the H-cluster is not generally affected by the mutation. Significant differences were observed only in the inhibited state Hox-CO: the vibrational modes assigned to the COexo and Fed-CO in this state are shifted to lower values in C298D, suggesting different interaction of these ligands with the protein moiety when C298 is changed to D298. More relevant to the catalytic cycle, the redox equilibrium between the Hox and Hred states is modified by the mutation, causing a prevalence of the oxidized state. This work highlights how the interactions between the protein environment and the H-cluster, a dynamic closely interconnected system, can be engineered and studied in the perspective of designing bio-inspired catalysts and mimics.},
doi = {10.1016/j.bbabio.2015.10.005},
url = {https://www.osti.gov/biblio/1234222}, journal = {Biochimica et Biophysica Acta - Bioenergetics},
issn = {0005-2728},
number = 1,
volume = 1857,
place = {United States},
year = {2016},
month = {1}
}