Dimerization of assimilatory NADPH-dependent sulfite reductase reveals elements for diflavin reductase binding at a minimal interface.

Escherichia coli NADPH-dependent assimilatory sulfite reductase is responsible for fixing sulfur for incorporation into sulfur-containing biomolecules. The oxidoreductase is composed of two subunits, an NADPH, FMN, and FAD-binding diflavin reductase and an iron siroheme and Fe 4 S 4 -containing oxidase. How they interact has been an unknown for over 50 years because the complex is highly flexible, thus has been intransigent for traditional X-ray or cryo-EM structural analysis. Using a combination of the chameleon plunging system with a fluorinated lipid we overcame the challenge of preserving the minimal dimer between the subunits for high-resolution cryo-EM analysis. Here, we report the first structure of the complex between the reductase and oxidase, revealing how they interact in a minimal interface. Further, we determined the structural elements that discriminate between the pairing of a siroheme-containing oxidase with a diflavin reductase or a ferredoxin partner to channel the six electrons that reduce sulfite to sulfide. Significance Statement: Sulfur is one of the essential building blocks of life. Sulfur exists in numerous redox states but only one can be incorporated into biomass - S 2- (sulfide). In Escherichia coli , a protein enzyme called sulfite reductase reduces sulfite by six electrons to make sulfide. Typical electron transfer reactions move one or two electrons at a time, so this chemistry is unique. To do so, E. coli uses a two protein complex with unique co-enzymes. To date, how the subunits interact so the co-enzymes can transfer electrons has remained a mystery because the complex is structurally dynamic, thus difficult to analyze with traditional methods. This study shows for the first time the structure of the enzyme complex that performs this unique chemistry.

A Diverged Transcriptional Network for Usage of Two Fe-S Cluster Biogenesis Machineries in the Delta-Proteobacterium Myxococcus xanthus.

Myxococcus xanthus possesses two Fe-S cluster biogenesis machineries, ISC (iron-sulfur cluster) and SUF (sulfur mobilization). Here, we show that in comparison to the phylogenetically distant Enterobacteria, which also have both machineries, M. xanthus evolved an independent transcriptional scheme to coordinately regulate the expression of these machineries. This transcriptional response is directed by RisR, which we show to belong to a phylogenetically distant and biochemically distinct subgroup of the Rrf2 transcription factor family, in comparison to IscR that regulates the isc and suf operons in Enterobacteria. We report that RisR harbors an Fe-S cluster and that holo-RisR acts as a repressor of both the isc and suf operons, in contrast to Escherichia coli, where holo-IscR represses the isc operon whereas apo-IscR activates the suf operon. In addition, we establish that the nature of the cluster and the DNA binding sites of RisR, in the isc and suf operons, diverge from those of IscR. We further show that in M. xanthus, the two machineries appear to be fully interchangeable in maintaining housekeeping levels of Fe-S cluster biogenesis and in synthesizing the Fe-S cluster for their common regulator, RisR. We also demonstrate that in response to oxidative stress and iron limitation, transcriptional upregulation of the M. xanthus isc and suf operons was mediated solely by RisR and that the contribution of the SUF machinery was greater than the ISC machinery. Altogether, these findings shed light on the diversity of homeostatic mechanisms exploited by bacteria to coordinately use two Fe-S cluster biogenesis machineries. IMPORTANCE Fe-S proteins are ubiquitous and control a wide variety of key biological processes; therefore, maintaining Fe-S cluster homeostasis is an essential task for all organisms. Here, we provide the first example of how a bacterium from the Deltaproteobacteria branch coordinates expression of two Fe-S cluster biogenesis machineries. The results revealed a new model of coordination, highlighting the unique and common features that have independently emerged in phylogenetically distant bacteria to maintain Fe-S cluster homeostasis in response to environmental changes. Regulation is orchestrated by a previously uncharacterized transcriptional regulator, RisR, belonging to the Rrf2 superfamily, whose members are known to sense diverse environmental stresses frequently encountered by bacteria. Understanding how M. xanthus maintains Fe-S cluster homeostasis via RisR regulation revealed a strategy reflective of the aerobic lifestyle of this organsim. This new knowledge also paves the way to improve production of Fe-S-dependent secondary metabolites using M. xanthus as a chassis.

The Siroheme-[4Fe-4S] Coupled Center.

In nature, sulfur exists in a range of oxidation states and the two-electron reduced form is the most commonly found in biomolecules like the sulfur-containing amino acids cysteine and methionine, some cofactors, and polysaccharides. Sulfur is reduced through two pathways: dissimilation, where sulfite (SO2-3) is used as terminal electron acceptor; and assimilation, where sulfite is reduced to sulfide (S2-) for incorporation into biomass. The pathways are independent, but share the sulfite reductase function, in which a single enzyme reduces sulfite by six electrons to make sulfide. With few exceptions, sulfite reductases from either pathway are iron metalloenzymes with structurally diverse configurations that range from monomers to tetramers. The hallmark of sulfite reductase is its catalytic center made of an iron-containing porphyrinoid called siroheme that is covalently coupled to a [4Fe-4S] cluster through a shared cysteine ligand. The substrate evolves through a push-pull mechanism, where electron transfer is coupled to three dehydration steps. Siroheme is an isobacteriochlorin that is more readily oxidized than protoporphyin IX-derived hemes. It is synthesized from uroporphyrinogen III in three steps (methylation, a dehydrogenation, and ferrochelation) that are performed by enzymes with homology to those involved in cobalamin synthesis. Future research will need to address how the siroheme-[4Fe-4S] clusters are assembled into apo-sulfite and nitrite reductases. The chapter will discuss how environmental microbes use sulfite reductase to survive in a range of ecosystems; how atomic-resolution structures of dissimilatory and assimilatory sulfite reductases reveal their ancient homology; how the siroheme-[4Fe-4S] cluster active site catalyzes the six-electron reduction of sulfite to sulfide; and how siroheme is synthesized across diverse microrganisms.

NADPH-dependent sulfite reductase flavoprotein adopts an extended conformation unique to this diflavin reductase.

This is the first X-ray crystal structure of the monomeric form of sulfite reductase (SiR) flavoprotein (SiRFP-60) that shows the relationship between its major domains in an extended position not seen before in any homologous diflavin reductases. Small angle neutron scattering confirms this novel domain orientation also occurs in solution. Activity measurements of SiR and SiRFP variants allow us to propose a novel mechanism for electron transfer from the SiRFP reductase subunit to its oxidase metalloenzyme partner that, together, make up the SiR holoenzyme. Specifically, we propose that SiR performs its 6-electron reduction via intramolecular or intermolecular electron transfer. Our model explains both the significance of the stoichiometric mismatch between reductase and oxidase subunits in the holoenzyme and how SiR can handle such a large volume electron reduction reaction that is at the heart of the sulfur bio-geo cycle.

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

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.

The DEAD-box Protein Rok1 Orchestrates 40S and 60S Ribosome Assembly by Promoting the Release of Rrp5 from Pre-40S Ribosomes to Allow for 60S Maturation.

DEAD-box proteins are ubiquitous regulators of RNA biology. While commonly dubbed "helicases," their activities also include duplex annealing, adenosine triphosphate (ATP)-dependent RNA binding, and RNA-protein complex remodeling. Rok1, an essential DEAD-box protein, and its cofactor Rrp5 are required for ribosome assembly. Here, we use in vivo and in vitro biochemical analyses to demonstrate that ATP-bound Rok1, but not adenosine diphosphate (ADP)-bound Rok1, stabilizes Rrp5 binding to 40S ribosomes. Interconversion between these two forms by ATP hydrolysis is required for release of Rrp5 from pre-40S ribosomes in vivo, thereby allowing Rrp5 to carry out its role in 60S subunit assembly. Furthermore, our data also strongly suggest that the previously described accumulation of snR30 upon Rok1 inactivation arises because Rrp5 release is blocked and implicate a previously undescribed interaction between Rrp5 and the DEAD-box protein Has1 in mediating snR30 accumulation when Rrp5 release from pre-40S subunits is blocked.

The N-terminal Domain of Escherichia coli Assimilatory NADPH-Sulfite Reductase Hemoprotein Is an Oligomerization Domain That Mediates Holoenzyme Assembly.

Assimilatory NADPH-sulfite reductase (SiR) from Escherichia coli is a structurally complex oxidoreductase that catalyzes the six-electron reduction of sulfite to sulfide. Two subunits, one a flavin-binding flavoprotein (SiRFP, the α subunit) and the other an iron-containing hemoprotein (SiRHP, the ß subunit), assemble to make a holoenzyme of about 800 kDa. How the two subunits assemble is not known. The iron-rich cofactors in SiRHP are unique because they are a covalent arrangement of a Fe4S4 cluster attached through a cysteine ligand to an iron-containing porphyrinoid called siroheme. The link between cofactor biogenesis and SiR stability is also ill-defined. By use of hydrogen/deuterium exchange and biochemical analysis, we show that the α8ß4 SiR holoenzyme assembles through the N terminus of SiRHP and the NADPH binding domain of SiRFP. By use of small angle x-ray scattering, we explore the structure of the SiRHP N-terminal oligomerization domain. We also report a novel form of the hemoprotein that occurs in the absence of its cofactors. Apo-SiRHP forms a homotetramer, also dependent on its N terminus, that is unable to assemble with SiRFP. From these results, we propose that homotetramerization of apo-SiRHP serves as a quality control mechanism to prevent formation of inactive holoenzyme in the case of limiting cellular siroheme.