Escherichia coli NADPH-dependent assimilatory sulfite reductase (SiR) reduces sulfite by six electrons to make sulfide for incorporation into sulfur-containing biomolecules. SiR has two subunits: an NADPH, FMN, and FAD-binding diflavin flavoprotein and a siroheme/Fe 4 S 4 cluster-containing hemoprotein. The molecular interactions that govern subunit binding have been unknown since the discovery of SiR over 50 years ago because SiR is flexible, thus has been intransigent for traditional high-resolution structural analysis. We used a combination of the chameleon plunging system with a fluorinated lipid to overcome the challenges of preserving a flexible molecule to determine a 2.78 Å-resolution cryo-EM structure of a dimeric complex between the subunits. chameleon, combined with the fluorinated lipid, overcame persistent denaturation at the air-water interface. Using a previously characterized minimal dimer between the subunits reduced the heterogeneity of a structurally heterogeneous complex to a level that could be analyzed using multi-conformer cryo-EM image analysis algorithms. Here, we report the first near-atomic resolution structure of the flavoprotein/hemoprotein complex, revealing how they interact in a minimal interface. Further, we determined the structural elements that discriminate between pairing a hemoprotein with a diflavin reductase, as in the E. coli homolog, or a ferredoxin partner, as in maize ( Zea mays ).
Significance statement: Sulfur is one of the essential building blocks of life. Sulfur exists in numerous redox states but only one can be incorporated into biomass - S 2- (sulfide). In Escherichia coli , a protein enzyme called sulfite reductase reduces sulfite by six electrons to make sulfide. Typical electron transfer reactions move one or two electrons at a time, so this chemistry is unique. E. coli SiR is a two-protein complex composed of a diflavin reductase flavoprotein and an iron metalloenzyme hemoprotein. Until now, the molecular interactions that govern subunit interactions remained a mystery because the extreme flexibility of the flavoprotein subunit, which has challenged X-ray or cryo-EM analysis for over 30 years. In overcoming these challenges, we used a combination of rapid plunging with a high critical-micelle-concentration lipid alongside a biochemically minimized complex to determine the 2.78 Å-resolution cryo-EM structure of a dimer between the flavoprotein and hemoprotein subunits.