Structure of dimerized assimilatory NADPH-dependent sulfite reductase reveals the minimal interface for diflavin reductase binding.

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/FeS 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 homolog, or a ferredoxin partner, as in maize ().