Assimilatory sulfite reductase (SiR) contains eight flavin-containing (SiRFP) and four iron-containing (SiRHP) subunits. Its conformational flexibility enables electron flow but hinders structural visualization. We took two SiRFP centered routes to study these phenomena: (1) disrupt the N-terminus responsible for SiRFP oligomerization through amino acid substitution and (2) trim the SiRFP internal linker (residues 211 to 239) to bias toward a more open state and test if intramolecular FAD to FMN transfer is preserved First, we examined the full-length SiRFP octamer, internal linker-truncated octamer, and a variant of the N terminus by tandem -trapped ion mobility mass spectrometry (t TIMS/MS) to learn about the native oligomeric state of the SiRFP assembly. Collision cross sections were taken at matched charge states and compared with SEC-MALS, AUC, and SANS. Next, we used in vitro activity assays to analyze the functional effect of the internal linker deletions on SiR activity. t TIMS analysis supports the longstanding hypothesis that SiRFP is, indeed, an octamer that can be disrupted by altering its N terminus (Q22A, Y39A, F40A, Q47A, SiRFP₄mut). SiRFP₄mut is primarily a monomeric species with minor contributions from dimeric, trimeric, and tetrameric assemblies. SEC-MALS and AUC agreed with this mixture, while SANS analysis is dominated by a tetramer, suggesting the solution scattering could be influenced by disorder features of this region. Nevertheless, SiRFP₄mut can still complement a SiRFP-deficient strain of Escherichia coli. Similar analysis of the internal linker shows significant resiliency: the complex appears to retain activity until the entire linker region is deleted. In conclusion, t TIMS provides sensitive fingerprints of SiRFP shape and stoichiometry. Linker truncating produces small compaction with increased charge sensitivity, and the nominal tetramer is not a single, well-defined assembly.
Ahmadizadeh et al. (Sun,) studied this question.