The Structure and Function of Sulfite Reductase Flavoprotein: Three Faces of a Dynamic Enzyme

Abstract

In enterobacteria like Escherichia coli, NADPH‐dependent assimilatory Sulfite Reductase (SiR) is the central enzyme in the sulfur assimilation pathway. SiR performs the six‐electron reduction of sulfite (SO3−2) to sulfide (S−2). SiR is a soluble multimeric enzyme with two subunits that combine in a 2:1 ratio to form a stable dodecameric complex. The α subunit (SiRFP) is a cytochrome P450 oxidoreductase (CYPOR) homolog. The β subunit (SiRHP) is a monomeric metalloenzyme with a Fe4S4 cluster and a porphyrinoid group called siroheme that forms the active site for the complex. SiRFP has the prototypical, three‐domain architecture of CYPOR that transfers electrons from the cellular donor NADPH to its FAD and FMN cofactors sequentially, ultimately delivering them to the binding partner, in this case SiRHP. However, SiRFP differs from other CYPORs in several ways: 1) SiRFP is a soluble protein that forms a homo‐octamer when expressed in the absence of SiRHP and 2) SiRFP forms a stable complex with SiRHP. The main aim of our research is to understand the electron transfer between the subunits in the context of the unique stoichiometry and structure of the complex. We identified two regions of SiRFP whose solvent accessibility changes upon assembly with SiRHP. The two regions of the structure are on different faces of the protein: one is in the N‐terminal FMN‐binding domain and the other is in the C‐terminal NADPH binding domain. Electrons are known to pass from the FMN binding domain to SiRHP. Interestingly, the interface that is part of the NADPH binding domain is sufficient to form a tight complex with SiRHP, suggesting the interface within the FMN binding domain is transient. To test this hypothesis, we synthesized three point variants: Y101A, Q191K, and V500D. Y101 is part of the FMN binding domain. Q191 plays a role in the conformational change that is known to govern electron transfer in CYPOR. V500 is part of the SiRFP region that interacts with SiRHP. Then, we used complementation assays and isothermal titration calorimetry (ITC) to probe the effect of these mutations. The three combined mutations has a synergic effect, resulting in cells that were unable to grow in minimal media. The ITC data showed that only the SiRFP V500D and variants combined with it had a reduced affinity for SiRHP respect to the wild‐type enzyme. From these results we conclude that SiR complex formation is driven by hydrophobic interactions in the C‐terminus of SiRFP, far from the site of electron transfer to its SiRHP partner. In contrast, transient interactions involving Y101 are important for electron transfer from the FMN cofactor to the metallic cofactors in SiRHP.Support or Funding InformationMCB1149763