Cyanophenylalanine as an Infrared Probe for Iron-Sulfur Cluster Redox State in Multi-Centre Metalloenzymes.

We demonstrate that 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. We observe a redshift of ≈ 1 - 2 cm-1 in the NC stretching frequency following reduction of spinach ferredoxin modified to contain CNF close to its [2Fe-2S] centre, 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, we observe a reversible shift in wavenumber for CNF 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 general applicability of this approach to studying complex metalloenzymes.