TY - JOUR
T1 - Cyanophenylalanine as an infrared probe for iron–sulfur cluster redox state in multicenter metalloenzymes
AU - Duan, Zehui
AU - Wei, Jiaao
AU - Carr, Stephen B.
AU - Ramirez, Miguel
AU - Evans, Rhiannon M.
AU - Ash, Philip A.
AU - Rodriguez-Macia, Patricia
AU - Sachdeva, Amit
AU - Vincent, Kylie A.
N1 - 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.
PY - 2025/7/18
Y1 - 2025/7/18
N2 - 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.
AB - 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.
U2 - 10.1002/cbic.202500251
DO - 10.1002/cbic.202500251
M3 - Article
SN - 1439-4227
VL - 26
JO - ChemBioChem
JF - ChemBioChem
IS - 14
M1 - e202500251
ER -