The effect of a C298D mutation in CaHydA [FeFe]-hydrogenase: Insights into the protein-metal cluster interaction by EPR and FTIR spectroscopic investigation

Morra, Simone; Maurelli, Sara; Chiesa, Mario; Mulder, David W.; Ratzloff, Michael W.; Giamello, Elio; King, Paul W.; Gilardi, Gianfranco; Valetti, Francesca

Abstract

© 2015 Elsevier B.V. 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 causes 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.

Citation

Morra, S., Maurelli, S., Chiesa, M., Mulder, D. W., Ratzloff, M. W., Giamello, E., …Valetti, F. (2016). The effect of a C298D mutation in CaHydA [FeFe]-hydrogenase: Insights into the protein-metal cluster interaction by EPR and FTIR spectroscopic investigation. BBA - Bioenergetics, 1857(1), 98-106. https://doi.org/10.1016/j.bbabio.2015.10.005

• Journal Article Type: Article
• Acceptance Date: Oct 15, 2015
• Online Publication Date: Oct 19, 2015
• Publication Date: 2016-01
• Deposit Date: Jan 31, 2020
• Publicly Available Date: Apr 1, 2020
• Journal: Biochimica et Biophysica Acta (BBA) - Bioenergetics
• Print ISSN: 0005-2728
• Electronic ISSN: 1879-2650
• Publisher: Elsevier
• Peer Reviewed: Peer Reviewed
• Volume: 1857
• Issue: 1
• Pages: 98-106
• DOI: https://doi.org/10.1016/j.bbabio.2015.10.005
• Public URL: https://nottingham-repository.worktribe.com/output/3841227
• Publisher URL: https://www.sciencedirect.com/science/article/pii/S0005272815002121
• Additional Information: This article is maintained by: Elsevier; Article Title: The effect of a C298D mutation in CaHydA [FeFe]-hydrogenase: Insights into the protein-metal cluster interaction by EPR and FTIR spectroscopic investigation; Journal Title: Biochimica et Biophysica Acta (BBA) - Bioenergetics; CrossRef DOI link to publisher maintained version: https://doi.org/10.1016/j.bbabio.2015.10.005; Content Type: article; Copyright: Copyright © 2015 Elsevier B.V. Published by Elsevier B.V. All rights reserved.