Cyanophenylalanine as an Infrared Probe for Iron–Sulfur Cluster Redox State in Multicenter Metalloenzymes

Journal article

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.

Authors: Duan, Z; Wei, J; Carr, SB; Ramirez, M; Evans, RM

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: Wiley
• Journal: ChemBioChem
• Article number: 2500251
• Publication date: 2025-05-26
• Acceptance date: 2025-05-09
• DOI: 10.1002/cbic.202500251
• EISSN: 1439-7633
• ISSN: 1439-4227
• Language: English
• Keywords: spectroelectrochemistry; iron–sulfur clusters; hydrogenase; cyanophenylalanine; infrared labels