Probing the Ferredoxin:Hydrogenase Electron Transfer Complex by Infrared Difference Spectroscopy

Ferredoxins are small iron-sulfur proteins that engage in electron transfer (ET) with oxidoreductases across all domains of life. In bacteria, ferredoxins that contain two [4Fe-4S] clusters differ with respect to their electric midpoint potential: while “Alvin”-type ferredoxins show individual potentials between -500 and -650 mV vs. SHE, clostridial ferredoxins perform ET at indistinguishable potentials of approximately -400 mV vs. SHE. In this work, the electron transfer complex between clostridial ferredoxin CpFd and [FeFe]-hydrogenases from Clostridium pasteurianum (CpI) and green algae Chlamydomonas reinhardtii (CrHydA) was investigated spectroscopically. [FeFe]-hydrogenases are oxidoreductases that catalyze hydrogen turnover in bacteria and algae. Introducing the non-canonical amino acid para-cyanophenylalanine (pCNF) near to one of the iron-sulfur clusters of CpFd allowed for a quantification of electric field changes via the vibrational Stark effect (VSE) by Fourier-transform infrared (FTIR) spectroscopy. Upon reduction with H2 or auto-oxidation under N2, in situ FTIR difference spectroscopy reports on protein structural changes. Our data reveal that the affinity between ferredoxin and its redox partners is modulated by redox-dependent protein-protein interactions (PPIs). Prompted by these findings, we discuss whether clostridial ferredoxins might act as two-electron redox partners in contact with hydrogenase or other oxidoreductases.