Investigation of Protein-Protein Interactions of Ferredoxin and Sulfite Reductase Under Different Sodium Chloride Concentrations by NMR Spectroscopy and Isothermal Titration Calorimetry

Sulfite reductase (SiR) catalyzes the reduction of sulfite to sulfide by using six electrons transported from ferredoxin (Fd) for eventual sulfur assimilation. As efficient electron flows are ensured by forming a productive Fd:SiR complex, detailed characterization of a Fd:SiR complex in solution is of particular importance. Here, we show that acidic residues of Fd play essential roles in forming an electron transfer complex with SiR by using attractive electrostatic interactions with putative basic residues of SiR. The thermodynamic approach using calorimetry revealed a favorable electrostatic contribution to form the Fd:SiR complex at the molecular level. Solution-state nuclear magnetic resonance (NMR) spectroscopy on 15N-labeled Fd in the presence of SiR showed large perturbations in NMR signals of acidic residues of Fd. The addition of NaCl diminished overall perturbations of NMR signals of SiR-bound Fd which resulted from the decrease in interprotein affinity. However, acidic residues at both termini still showed relatively large peak perturbation. These results at the residue level suggested that intermolecular interactions between Fd and SiR are electrostatic in nature and the electrostatic interaction is a dominant contributor to form the Fd:SiR complex. We suggest that a combination of calorimetry and NMR is a powerful approach to investigating protein-protein interactions.