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

Abstract

Ferredoxins are small iron-sulfur proteins that engage in one-electron transfer with oxidoreductases across all domains of life. The catalyzed reactions often include multiple electrons, e.g., in the two-electron reduction of NADP+ during photosynthesis or the reduction of protons to H2 by the metalloenzyme hydrogenase. To date, the microscopic details of how ferredoxins facilitate multiple electron redox chemistry are unknown. Ferredoxins of the Allochromatium vinosum subfamily contain two [4Fe-4S] clusters, which allows for two one-electron transfer reactions. However, the iron-sulfur clusters of 2[4Fe-4S]-type ferredoxins typically have very similar reduction potentials and conclusive evidence for the transfer of two electrons during a single protein-protein interaction (PPI) has not been reported. In this work, the electron transfer complexes between clostridial 2[4Fe-4S] ferredoxin, CpFd, and [FeFe]-hydrogenases from both Clostridium pasteurianum (CpI) and Chlamydomonas reinhardtii (CrHydA) were investigated. Introducing a non-canonical amino acid near to one of the iron-sulfur clusters of CpFd permitted the quantification of electric field changes via the vibrational Stark effect by Fourier-transform infrared (FTIR) spectroscopy. Upon reduction, in situ FTIR difference spectroscopy reported on protein structural changes and microscale thermophoresis revealed that the affinity between ferredoxin and hydrogenase is modulated by redox-dependent PPIs. Prompted by these findings, we suggest a model how ferredoxin efficiently facilitates multiple electron redox chemistry based on individual one-electron transfer reactions.