Assimilatory NADPH-dependent sulfite reductase (SiR) is the enzyme responsible for the six-electron reduction of sulfite to sulfide. In Enterobacteria, SiR is a dodecameric complex with two subunits: an octameric flavin-containing SiRFP, and four copies of a monomeric iron-containing subunit (SiRHP). SiRFP is a homologous to cytochrome P-450 reductase (CYP), each of which has three main domains: NADPH/FAD-binding domain, FMN-binding domain and a connecting domain that is responsible of the relative orientation of the other two domains. However, SiRFP differs from CYP because SiRFP is a soluble protein that forms an octamer through its first 51 residues and is a stable complex with the oxidase subunit. SiRHP has a Fe4S4 cluster and a siroheme cofactor in its active site. In the SiR holoenzyme electrons flow from NADPH to FAD to FMN in SiRFP and from the FMN cofactor to SiRHP. SiRFP, SiRHP, and PAPS sulfotransferase, another enzyme required for sulfate assimilation, are encoded by three genes in the cysJIH operon, therefore, they can be independently expressed and purified. In this way, each subunit has been characterized structural and functionally, however the structure of the SiR holoenzyme remains ill defined. Therefore, the main aim of this dissertation is to understand SiR assembly and the mechanism of electron transfer between subunits. I made important advances in understanding SiR’s subunit interactions by the use of a variety of biochemical and structural techniques. First, we confirmed the stoichiometry of the holoenzyme as an α8β4 complex. I further discovered that in this complex, SiRFP has two interfaces with SiRHP. The Region 1, is in SiRFP’s C-terminus and it is responsible for forming the stable interaction between each SiR subunits. Region 2 is in SiRFP’s N-terminus and we hypothesize that it is involved in a transient interaction with SiRHP and, such as, is the one required for electron transfer between subunits. My more detailed characterization shows that complex formation depends on hydrophobic interactions between the subunits. In further characterization this interaction, I identified three regions of predicted intrinsically disorder/disorder-based binding sites in each subunit that are required for SiR assemble and function. First, the N-terminus of SiRHP has characteristics of a disorder-base binding region, and it is required for holoenzyme complex formation. Second, the N-terminus of SiRFP is required for octamerization of this subunit. Third, the linker between the FMN-binding domain and FAD/NADPH binding domain is also an intrinsically disorder region that orients the FMN-domain relative to rest of the complex. As result of my experiments, I propose a model for electron transfer in which all SiRFP subunits participate in electron transfer to SiRHP. This model explains the high efficiency of the complex that is observed by the absence of partially reduced sulfur-oxygen intermediates. Additionally, advances were made to prepare cryo-electron microscopy samples of SiR based in the new insights into SiR’s structural characteristics. Additionally, a new reductionist approach using x-ray crystallography is proposed to better study SiR subunits interactions.
