Structure–Function Relationships in the Oligomeric NADPH-Dependent Assimilatory Sulfite Reductase

Abstract

The central step in the assimilation of sulfur is a six-electron reduction of sulfite to sulfide, catalyzed by the oxidoreductase NADPH-dependent assimilatory sulfite reductase (SiR). SiR is composed of two subunits. One is a multidomain flavin binding reductase (SiRFP) and the other an iron-containing oxidase (SiRHP). Both enzymes are primarily globular, as expected from their functions as redox enzymes. Consequently, we know a fair amount about their structures but not how they assemble. Curiously, both structures have conspicuous regions that are structurally undefined, leaving questions about their functions and raising the possibility that they are critical in forming the larger complex. Here, we used ultraviolet-visible and circular dichroism spectroscopy, isothermal titration calorimetry, proteolytic sensitivity tests, electrospray ionization mass spectrometry, and activity assays to explore the effect of altering specific amino acids in SiRFP on their function in the holoenzyme complex. Additionally, we used computational analysis to predict the propensity for intrinsic disorder within both subunits and found that SiRHP's N-terminus is predicted to have properties associated with intrinsic disorder. Both proteins also contained internal regions with properties indicative of intrinsic disorder. We showed that SiRHP's N-terminal disordered region is critical for complex formation. Together with our analysis of SiRFP amino acid variants, we show how molecular interactions outside the core of each SiR globular enzyme drive complex assembly of this prototypical oxidoreductase.

Small-angle neutron scattering solution structures of NADPH-dependent sulfite reductase

“…In our model for the positions of SiRFP-60 and SiRHP within the envelopes modeled from neutron scattering, SiRHP is far from the Fld domain that would be responsible for funneling electrons to it within this minimal dimer (Figure 3D). This placement is consistent with the results from contrast matching experiments (Figures 4 and 5) as well as previously-reported mutational analysis that identified amino acids in the FNR domain of SiRFP as important for SiRHP binding (Askenasy et al ., 2018; Askenasy et al ., 2015). Nevertheless, cis electron transfer to a tightly-bound SiRHP in the minimal dimeric complex occurs, albeit at a rate of ∼50% that of the holoenzyme (Tavolieri et al ., 2019; Zeghouf et al ., 2000).…”

“…Although structures of the monomeric SiR subunits are known from X-ray crystallography (Crane et al ., 1995; Gruez et al ., 2000; Tavolieri et al ., 2019), no structures exist for higher-order complexes. Consequently, we do not know how the subunits interact such that there is a tight-binding, structural interface independent of the transient, functional interface that mediates electron transfer (Askenasy et al ., 2018; Askenasy et al ., 2015). Additionally, we do not know if subunit assembly affects the relative domain orientations of the conformationally dynamic SiRFP.…”

“…The 8:4 stoichiometry of reductase to oxidase subunits in SiR has been long-hypothesized as important in its high-volume electron transfer chemistry by increasing the local concentration of reduced Fld domains for each SiRHP active site (Askenasy et al ., 2018; Siegel & Davis, 1974; Tavolieri et al ., 2019). This hypothesis predicts that SiRHP can recruit a transiently-interacting Fld domain from either a tightly-bound subunit (cis electron transfer) or an adjacent partner ( trans electron transfer) (Figure 1D).…”

“…SiRHP’s D max decreases by almost 10 Å upon binding its partner, which can be visualized in the envelope function as a transition to a more globular shape. This compaction is likely the result of a rearrangement at its N-terminus, which is not resolved in the crystal structure, but known to be required for SiRFP binding (Askenasy et al ., 2018; Crane et al ., 1995). We propose that binding to SiRFP’s FNR domain either induces the flexible N-terminus to contract as it mediates intersubunit contacts or binds SiRFP as an extended peptide that would not contribute strongly to SiRHP’s scattering.…”

“…Another aspect of SiR that sets it apart from analogous systems is the way in which SiRFP and SiRHP interact, through interactions between SiRHP’s N-terminus and a position on SiRFP’s FNR domain that is far from the Fld domain (Askenasy et al , 2018; Askenasy et al , 2015). This interaction is counterintuitive because the Fld domain must interact with SiRHP to pass electrons to its siroheme-Fe 4 S 4 cofactors that funnel the electrons to the siroheme-bound substrate.…”