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

## TL;DR

Cyanophenylalanine is used as a tool to study electron transport in metalloenzymes by detecting changes in iron-sulfur cluster redox states through infrared spectroscopy.

## Contribution

Cyanophenylalanine is shown to be a general and reversible infrared probe for monitoring redox states of iron-sulfur clusters in complex metalloenzymes.

## Key findings

- Cyanophenylalanine shows a reversible redshift in nitrile stretching frequency upon reduction of iron-sulfur clusters in spinach ferredoxin.
- The same redshift is observed in [FeFe] hydrogenase with proximal [4Fe–4S] clusters, demonstrating applicability to different metalloenzymes.
- The method works for both apo- and holo-proteins, indicating its broad utility in studying electron transport chains.

## 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.

The noncanonical amino acid, cyanophenylalanine, is genetically inserted close to iron–sulfur clusters of either spinach ferredoxin or iron–iron hydrogenase, and functions as an infrared spectroscopic reporter for changes in cluster redox state.© 2025 WILEY‐VCH GmbH