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

Highlights

• Sulfite reductase (SiR) is a multi-subunit oxidoreductase that reduces SO₃²⁻ to S²⁻.

• We show that subunit-subunit binding triggers domain reorganization of the reductase subunit.

• Subunit-subunit binding also elicits compaction of the oxidase subunit.

• Reducing the reductase positions its electron-transfer domain near the oxidase binding site.

• These domain motions present a model for multi-electron electron transfer in SiR.

Abstract

Sulfite reductase (SiR), a dodecameric complex of flavoprotein reductase subunits (SiRFP) and hemoprotein oxidase subunits (SiRHP), reduces sulfur for biomass incorporation. Electron transfer within SiR requires intra- and inter-subunit interactions that are mediated by the relative position of each protein, governed by flexible domain movements. Using small-angle neutron scattering, we report the first solution structures of SiR heterodimers containing a single copy of each subunit. These structures show how the subunits bind and how both subunit binding and oxidation state impact SiRFP’s conformation. Neutron contrast matching experiments on selectively deuterated heterodimers allow us to define the contribution of each subunit to the solution scattering. SiRHP binding induces a change in the position of SiRFP’s flavodoxin-like domain relative to its ferredoxin-NADP⁺ reductase domain while compacting SiRHP’s N-terminus. Reduction of SiRFP leads to a more open structure relative to its oxidized state, re-positioning SiRFP’s N-terminal flavodoxin-like domain towards the SiRHP binding position. These structures show, for the first time, how both SiRHP binding to, and reduction of, SiRFP positions SiRFP for electron transfer between the subunits.

Introduction

Multi-subunit oxidoreductase enzymes catalyze electron transfer reduction–oxidation (redox) reactions that drive energy flux in cells. A conserved class of NADPH-dependent diflavin reductases funnel electrons from NADPH through flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) cofactors to diverse oxidases that are often metalloenzymes. Assimilatory NADPH-dependent sulfite reductase (SiR) is a member of this class of enzymes and is responsible for the six-electron reduction of sulfite (SO₃²⁻) to sulfide (S²⁻) for incorporation into sulfur-containing biomolecules (Siegel et al., 1973). These SiRs are dodecameric, with a uniquely octameric diflavin reductase (SiRFP, the α subunit, Fig. 1a) and four copies of a siroheme- and Fe₄S₄-containing hemoprotein (SiRHP, the β subunit, Fig. 1b) (Murphy et al., 1973, Siegel and Davis, 1974, Siegel et al., 1974).

SiR’s α₈β₄ dodecameric assembly sets it apart from other members of this class of diflavin reductase-dependent enzymes like cytochrome P450 reductase (CPR), nitric oxide synthase (NOS), cytochrome P102, and methionine synthase (Campbell et al., 2014, Olteanu and Banerjee, 2001, Xia et al., 2011a, Zhang et al., 2018). Based on what is known about the well-studied CPR (Freeman et al., 2017, Freeman et al., 2018), electron transfer within the SiR diflavin reductase subunit is hypothesized to work through a redox-sensitive conformational change between two domains that are separated by a flexible hinge. Specifically, the FMN-binding domain, which is homologous to small flavodoxins (Fld), moves close to the NADPH-reduced FAD, which binds to a ferredoxin-NADP⁺ reductase (FNR) domain, to itself become reduced (Huang et al., 2013, Iyanagi et al., 2012, Wang et al., 1997). Upon NADP⁺ release, the Fld domain swivels back out to pass the electrons to its oxidase partner (Hamdane et al., 2009, Xia et al., 2011b). In CPR, the opening of the Fld domain creates the binding site for the heme-containing oxidase (Im and Waskell, 2011). In NOS, where the two subunits are expressed in a single polypeptide that dimerizes, this conformational change allows the reductase from one subunit to interact with the heme-binding oxidase domain from the partner (Campbell et al., 2014, Haque et al., 2018). The absence of any identified structure of the bound SiR subunits leaves a gap in our understanding of how intersubunit interactions and domain motions govern electron transfer in SiR.

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-Fe₄S₄ cofactors that funnel the electrons to the siroheme-bound substrate. Nevertheless, a separate, transient interaction between SiRFP’s Fld domain and a tightly-bound SiRHP is sufficient for SiR activity because a heterodimeric complex between a monomeric form of SiRFP and full-length SiRHP is active for SO₃²⁻ reduction, albeit at reduced efficiency (Gruez et al., 2000, Zeghouf et al., 2000). SiR’s capability for electron transfer is further affected if the flexible linker mediating domain movements of SiRFP is truncated, reducing activity in the heterodimer but not the dodecamer (Tavolieri et al., 2019). These results suggest a model in which the dodecamer transfers electrons through multiple pathways, first from an NADPH to the FAD and on to the FMN cofactor, either within a single SiRFP (intramolecular transfer) or from the FAD of one subunit to the FMN of an adjacent molecule (intermolecular transfer), and then either to a tightly bound SiRHP (in cis, Fig. 1c) or to a SiRHP on an adjacent SiRFP subunit (in trans, Fig. 1c). This redundancy in electron donor/acceptor pairing could explain SiR’s ability to catalyze high-volume electron transfer without releasing partially-reduced intermediates (Hsieh et al., 2010, Lancaster, 2018, Mirts et al., 2018, Oliveira et al., 2011).

In this study, we used small-angle neutron scattering (SANS), selective deuteration, solvent contrast variation, anaerobic reductions, and analytical ultracentrifugation (AUC) to probe the effects of altering the redox state and subunit binding on heterodimers of a monomeric SiRFP and its SiRHP partner. AUC on SiR heterodimers revealed sedimentation coefficients consistent with their respective oligomeric states as in vitro reconstituted specimen. SANS was used to measure hydrodynamic parameters and uncover the first-ever solution structures of these heterodimers. Through the use of selective deuteration and neutron contrast variation, the isolated scattering components of SiR heterodimers illuminated the contributions from each subunit to their complexes, informing our understanding of this multisubunit enzyme. Anaerobically reduced SiRFP variants were similarly measured by SANS and showed movements between the Fld and FNR domains. Together, these observations help explain the nature of its complex assembly as well as its capacity for electron transfer.