Duan Zehui, Wei Jiaao, Carr Stephen B, Ramirez Miguel, Evans Rhiannon M, Ash Philip A, Rodriguez-Macia Patricia, Sachdeva Amit, Vincent Kylie A
Department of Chemistry, University of Oxford, Inorganic Chemistry Laboratory, South Parks Road, Oxford, OX1 3QR, UK.
Research Complex at Harwell, Rutherford Appleton Laboratory, Harwell Campus, Didcot, OX11 0FA, UK.
Chembiochem. 2025 Jul 18;26(14):e202500251. doi: 10.1002/cbic.202500251. Epub 2025 May 26.
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 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]), 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.
非标准氨基酸对氰基苯丙氨酸（CNF）掺入金属蛋白后，可作为铁硫簇氧化还原状态的红外光谱探针，为确定复杂金属酶电子传递链中的电子占据情况提供了一种策略。在将菠菜铁氧化还原蛋白修饰为在其[2Fe-2S]中心附近含有CNF后，观察到腈（NC）伸缩频率发生了约1 - 2 cm的红移，并且这种位移在再氧化时会逆转。我们将此扩展到定位在脱硫脱硫弧菌[FeFe]氢化酶近端[4Fe-4S]簇附近的CNF。近端簇与远端[4Fe-4S]簇以及活性位点“H-簇”（[4Fe-4S]）的[4Fe-4S]簇相结合，形成了一个将活性位点与蛋白质表面相连的电子中继。同样，在脱辅基蛋白（含有铁硫簇但缺乏活性位点）或具有完整活性位点的全蛋白中簇还原后，观察到CNF波数的可逆位移，证明了该方法在研究复杂金属酶方面的普遍适用性。
