Structure-Function Analysis of the Multicomponent Redox Enzyme Sulfite Reductase
Murray, Daniel Travis (author)
Stroupe, M. Elizabeth (Margaret Elizabeth) (professor directing dissertation)
Li, Hong (university representative)
Jones, Kathryn M. (committee member)
Bass, Hank W. (committee member)
Tang, Hengli (committee member)
Florida State University (degree granting institution)
College of Arts and Sciences (degree granting college)
Department of Biological Science (degree granting department)
Escherichia coli NADPH-dependent assimilatory sulfite reductase, SiR, provides reduced sulfur for assimilation into biomass. The ability of SiR to perform its high-volume electron transfer reaction without the release of partially reduced sulfur-oxygen intermediates is hypothesized to be the result of its unique stoichiometry and inherent flexibility that enable a cis/trans electron transfer mechanism. SiR is comprised of an octameric flavoprotein, SiRFP, and four copies of a hemoprotein, SiRHP, that assemble in an 8:4 complex. NADPH donates electrons to SiRFP, where they are stored and transferred between domains before being transferred to SiRHP, the site of sulfur reduction. The flavin-binding domains of SiRFP, known as the FNR and Fld domains, are connected by a flexible linker, whereas the N-terminal domain is responsible for octamerization. Sulfite is reduced by six electrons before being released from SiRHP's active site as sulfide. How this metalloflavoprotein performs its reaction with such efficiency is incompletely understood. This dissertation analyzes the structure and function of SiR using a combination of structural, biochemical, and biophysical techniques in order to better understand this dynamic oxidoreductase. Thus, Chapter 1 serves as an introduction into what is currently known of SiR.Small-angle neutron scattering (SANS) features prominently throughout this dissertation and will be discussed at length in Chapter 2. This technique is used to elucidate the solution structures of SiR proteins, showing monomeric SiRFP to have an extended conformation as oxidized, counter to its diflavin reductase. The extended conformation of SiRFP is conducive to productive electron transfer to neighboring subunits in its higher-order complexes. Previous studies show that an internal truncation of SiRFP's flexible linker diminishes sulfur-reducing activity in SiR heterodimer but not in the holoenzyme. Anaerobic reduction assays on SiRFP variants are analyzed by UV-Visible spectrophotometry and show that its flavins are capable of being reduced by both dithionite and NADPH in the face of the internal truncation, which supports the notion that electron transfer may occur inter- and intramolecularly between SiRFP domains. These studies are described in detail throughout Chapter 3. In Chapter 4, SANS of reduced SiRFP monomer reveals a novel conformation, placing its Fld domain close to the binding interface with SiRHP. The transition between open and closed conformations by SiRFP upon reduction is not unprecedented, as demonstrated by its diflavin reductase homologs. Chapter 4 also includes neutron scattering studies that provide the first solution structure for a SiR heterodimer with assignments for the positions of each subunit and their domains. To confirm subunit and domain assignments within those structures, neutron contrast variation is used to further elucidate each subunit. In this approach, deuterium labeling of SiRHP, in vitro reconstitution of complexes with partially deuterated SiRHP and hydrogenated SiRFP subunits, and alteration of solvent H2O:D2O ratios allow for the manipulation of neutron contrast and the obtainment of isolated scattering profiles for each component of SiR. These experiments also show that SiRFP adopts an extended confirmation upon binding SiRHP and SiRHP becomes more compact. Structural details of higher-order assemblies of SiR, namely, octameric SiRFP and the dodecameric holoenzyme, have proven challenging to obtain. Chapter 5 presents the results of SANS measurements of these complexes, combined with contrast variation, that yielded the overall size, shape, and conformation of SiR in solution. Combined with analytical ultracentrifugation, the long-hypothesized stoichiometry of SiR is confirmed to be 8:4. The SiR dodecamer exhibits flexibility, adopting a heterogeneous ensemble of subunit orientations and domain conformations. SiRFP's structure undergoes a global compaction upon complex formation with SiRHP and SiRHP subunits that exist in a dynamic range of positions. SiRHP exists in peripheral locations about the complex as well as positions that may approach one another. The centers of mass of SiRFP and SiRHP are separated from one another in the holoenzyme, suggesting SiR is an asymmetric complex. Together, these results represent an initial mapping of SiR's structure and indicate a model for electron transfer in SiR that occurs in a cis and trans manner that may be further tested by studies informed upon the results described herein. Moving forward, as described in Chapter 6, SANS of anaerobically reduced SiR may reveal redox-sensitive structural rearrangements not immediately obvious from measurements of oxidized enzyme. Additionally, high-resolution structures of SiR may be obtained by affinity capture using streptavidin monolayer electron microscopy grids, where biotinylated SiRFP octamer or SiR dodecamer become immobilized on 2D streptavidin monolayer-crystals before their analysis with cryogenic-electron microscopy (cryo-EM).
Enzymology, Protein biochemistry, Small-angle scattering
October 22, 2021.
A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy.
Includes bibliographical references.
M. Elizabeth Stroupe, Professor Directing Dissertation; Hong Li, University Representative; Kathryn M. Jones, Committee Member; Hank Bass, Committee Member; Hengli Tang, Committee Member.
Florida State University