Cyanophenylalanine as an infrared probe for iron–sulfur cluster redox state in multicenter metalloenzymes

Duan, Zehui, Wei, Jiaao, Carr, Stephen B., Ramirez, Miguel, Evans, Rhiannon M., Ash, Philip A., Rodriguez-Macia, Patricia, Sachdeva, Amit and Vincent, Kylie A. (2025) Cyanophenylalanine as an infrared probe for iron–sulfur cluster redox state in multicenter metalloenzymes. ChemBioChem, 26 (14). ISSN 1439-4227

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Abstract

The noncanonical amino acid, para-cyanophenylalanine (CNF), when incorporated into metalloproteins, functions as an infrared spectroscopic probe for the redox state of iron-sulfur clusters, offering a strategy for determining electron occupancy in the electron transport chains of complex metalloenzymes. A redshift of ≈1–2 cm−1 in the nitrile (NC) stretching frequency is observed, following reduction of spinach ferredoxin modified to contain CNF close to its [2Fe–2S] center, and this shift is reversed on re-oxidation. We extend this to CNF positioned near to the proximal [4Fe–4S] cluster of the [FeFe] hydrogenase from Desulfovibrio desulfuricans. In combination with a distal [4Fe–4S] cluster and the [4Fe–4S] cluster of the active site ‘H-cluster’ ([4Fe–4S]H), the proximal cluster forms an electron relay connecting the active site to the surface of the protein. Again, a reversible shift in wavenumber for CNF is observed, following cluster reduction in either apo-protein (containing the iron-sulfur clusters but lacking the active site) or holo-protein with intact active site, demonstrating the general applicability of this approach to studying complex metalloenzymes.

Item Type:	Article
Additional Information:	Data Availability Statement: The data that support the findings of this study are available in the supplementary material of this article. Acknowledgements: Z.D. and J.W. contributed equally to this work. The work of K.A.V., P.A.A., R.M.E., and S.B.C. has been supported by the Biotechnology and Biological Sciences Research Council (BBSRC), grants BB/R018413/1 and BB/X002624/1) and European Research Council grant (ERC-2018-CoG BiocatSusChem 819580, which additionally supported work of M.R.). Work performed in A.S.'s laboratory was funded by the Wellcome Trust (204593/Z/16/Z), BBSRC (BB/R004692/1), and the University of East Anglia. P.A.A. is supported by BB/X002292/1 from the BBSRC. P.R.-M. has been supported by a Glasstone Fellowship and is grateful to Linacre College, Oxford, for a Junior Research Fellowship. J.W. was supported by a scholarship from the China Scholarship Council. The authors are grateful to Dr. James Birrell and Prof. Serena DeBeer (MPI-CEC) for kindly gifting us a plasmid encoding WT apo-DdHydAB. The authors are grateful to Dr. Will Myers and the CAESR suite, University of Oxford, for EPR measurements and analysis.
Faculty \ School:	Faculty of Science > School of Chemistry, Pharmacy and Pharmacology
UEA Research Groups:	Faculty of Science > Research Groups > Chemistry of Life Processes
Depositing User:	LivePure Connector
Date Deposited:	17 Sep 2025 16:30
Last Modified:	17 Sep 2025 19:30
URI:	https://ueaeprints.uea.ac.uk/id/eprint/100428
DOI:	10.1002/cbic.202500251

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