Understanding the Effects of Higher-Order Assembly on the Structure of Sulfite Reductase

Oxidoreductase enzyme complexes are one of the known biomolecular machines, which play a crucial role in cellular biochemistry including metabolic, signaling, and electron transport pathways. In these complexes, individual subunits can oligomerize to form homo-multimers or hetero-multimers and can transiently interact with redox enzyme partners to perform multistep enzymatic reactions. Multi-subunit oxidoreductases can show inter- and intra-subunit domain interactions by switching from open to closed conformations in order to deliver electrons to their oxidase partners. Assimilatory sulfite reductase (SiR) is an NADPH-dependent multidomain oxidoreductase that catalyzes sulfur reduction to produce sulfide, the bioavailable form of sulfur for subsequent synthesis of biomolecules. In Escherichia coli, SiR is a dodecameric holoenzyme, consisting of eight subunits of a flavoprotein (SiRFP) and four subunits of a hemoprotein (SiRHP). SiRFP is a diflavin reductase, a homolog of cytochrome P450 reductase (CPR) that is composed of the following domains: a FMN binding flavodoxin (Fld)-like-domain, a connection domain interrupting FAD/NADPH bind- ing FNR (Ferredoxin/NADP+ reductase)-like-domain and a flexible linker connecting Fld and FNR domains. This flexible linker is ∼ 30 amino acids long and remains crucial for transferring electrons from one domain to another. SiRHP is a metal-harboring domain that contains a siroheme (iron- containing tetrapyrrole) cofactor at the active site covalently coupled with Fe4S4 cluster through cysteine conjugations. Chapter 1 provides an introduction of multi domain oxidoreductases with a main focus on SiR and its homologs. Chapter 2 provides information about small-angle scattering technique including both neutron scattering and X-ray scattering. Small-angle neutron scattering (SANS) and neutron contrast variation technique are discussed in detail. SANS is used to analyze the solution structure of proteins and gives information about the radius of gyration, inter atomic distances between the particles, shape and size of the protein sample and also gives an approximate idea about the molecular weight of the sample. Chapter 3 includes the structural characterization of the SiR complex. SANS, neutron contrast variation, analytical ultracentrifugation and other biophysical techniques showed that SiR is an asymmetric complex. SiRFP undergoes compaction in the complex as compared to the free SiRFP and the four SiRHP binds at the periphery of the complex. This study showed the first solution structure of the SiR complex. Chapter 4 provides information about the two conformationally restricted variants of SiRFP and showed the possibility of open and closed conformations of SiRFP. This study showed the presence of domain crossover in SiRFP which indicates that the Fld domain of one polypeptide can interact with the FNR domain of an adjacent polypeptide. The SANS measurements provide the first model of the possible arrangement of eight subunits of SiRFP and showed that the N-terminal helical bundle acts as a central core of the complex. Chapter 5 presents a study about a novel variant of SiRFP which forms a tetramer. The N- terminus region of SiRFP consists of only 52 amino acids which oligomerizes to form an octamer. This study showed that four amino acids (Y39, F40, Q22, Q47) of the SiRFP's N-terminus region play a crucial role in the oligomerization of the eight copies of SiRFP. These residues show aromatic stacking (Y39, F40) and salt bridge interactions (Q22, Q47) between the helices and thus form a strong octameric bundle. Mutating these four amino acids results in a tetrameric assembly of SiRFP. The ultra stable properties of SiRFP's N-terminus octameric helical bundle are also discussed in this chapter. Chapter 6 includes the cryo-EM studies of SiR. SiR is a challenging sample to study using cryo-EM due to its sensitivity to the air-water interface during the grid preparation. This chapter reveals that a minimal binding interface is present between the SiRFP and SiRHP dimer. This study also shows the first high resolution structure of the SiRFP/SiRHP variant and explains that this minimal interaction is governed by the N-terminus of SiRHP and the FNR domain of SiRFP. Additionally, the highly flexible Fld domain of SiRFP explains its accessibility for transferring electrons to its redox partner. The last chapter highlights the future direction of the SiR project. It explains the ongoing crystallization related work for the different variants of SiRFP. Chapter 7 also includes the troubleshooting of SiR's sample preparation for cryo-EM studies which aims to solve the high resolution structure of this complex.