46449-B4
Mechanisms of Sulfur Reduction by Sulfur/Polysulfide Reductase
The focus of our work under this grant is to determine the detailed mechanism of sulfur/polysulfide reduction by polysulfide reductase at the molecular level. Our efforts are divided into 2 areas of emphasis: 1) studies of the polysulfide reductase (PsrABC) complex of Shewanella, and 2) detailed characterization of a recently discovered soluble polysulfide reductase present in S. loihica PV-4 and S. frigidimarina (not contained in the grant proposal and based on our recent discovery of a second polysulfide-reducing enzyme).
In the construction of the recombinant PsrABC complex, we have overexpressed and purified the membrane anchor subunit (psrC). We determined that the recombinant, soluble protein contains the expected bound quinone, indicating that the protein is in an active form (surprising given its in vivo role as an integral membrane protein). Some of this solubility may be derived from the thioredoxin tag present at the N-terminus of the protein during expression and purification, although the protein remains soluble after the thioredoxin (and poly-histidine) tag is cleaved from the protein or when expressed with only the poly-histidine tag present. The PsrB subunit has also been overexpressed and purified as a poly-histidine tagged protein, and shows UV-visible spectra consistent with the presence of the Fe-S centers observed previously. Exposure of PsrB to air, however, was shown to result in quantitative loss of iron, so we are currently purifying and working with this protein anaerobically.
PsrABC is a membrane complex, and in order to make characterization of the system easier one of our goals was to reconstitute the complex in a reusable film via electrostatic self-assembly. When the PsrC subunit was incubated with a polyanionic (polyethylimine) film, quartz crystal microbalance results indicated that psrC was absorbed into the film, essentially sinking down “into” the film in a manner analogous to the interaction of an integral membrane protein with a membrane. When PsrB was incubated with the PsrC-containing film, the results were consistent with PsrB binding on the surface of the film or to PsrC. Both PsrB and C were resistant to being washed off the film by buffer, and appeared to be essentially immobilized within or on the film. We confirming these results, and will include the PsrA subunit when it is purified as an active, soluble protein.
We are currently optimizing overexpression of the molybdopterin-containing PsrA subunit. The polyhistidine-tagged psrA is overexpressed in copious quantities; however, the protein precipitates during the overexpression. We are attempting to obtain soluble protein by several different methods, including 1) resolubilization in the presence of detergents followed by subsequent removal of the detergent, 2) anaerobic overexpression of the protein, (which has been shown to slow down overexpression), and 3) aerobic and anaerobic homologous overexpression of PsrA in the organism which it is naturally found in (Shewanella oneidensis MR-1). The advantage to the third method is that any proteins necessary for the assembly of the molybdopterin or iron-sulfur cofactors would be present, hopefully allowing for the production of soluble active protein. In addition, we have obtained a mutant of this species of Shewanella that does not contain the PsrA protein, allowing for site-directed mutagenesis of the protein in this system without having to worry about the wild-type PsrA background.
One of the major sticking points in our work has been the development of a reliable, accurate and precise assay for sulfide production. While a methylene-blue generating assay is used extensively throughout the literature, in our exhaustive attempts to work with this assay we have found it to be completely unreliable for the precision kinetic assays we wish to perform. We have therefore adapted an assay using the formation of CuS colloids and a sulfide-sensitive electrode for our measurement of sulfide produced by the complex. We have also developed the necessary assay for determination of the reverse reaction of the enzyme (oxidation of sulfide with the subsequent reduction of a quinone-based dye), so that when active PsrA protein is obtained are in an excellent position to quickly progress with our mechanistic work.
In a departure from the precise work defined in our proposal, while surveying Shewanella genomes we noticed that S. loihica PV-4 contained a homologue to a family of flavoproteins that another laboratory had demonstrated to be involved in the reduction of polysulfide. We have overexpressed and purified this FAD-dependent NADH-dependent and coenzymeA-activated polysulfide reductase. This is an entirely new enzymatic reaction, since the previously observed reaction with the Pyrococcus homologue was sulfur reduction in a CoA-dependent manner, with NADH substrate showing an extremely high Km (5 mM), while the reaction we’ve observed is the reduction of polysulfide in a CoA-activated manner, with a low mmolar Km for NADH. We are currently characterizing the mechanism of this enzyme using site-directed mutagenesis and steady and presteady-state kinetic techniques, and determining the role of a cysteine-containing “tail” unique to the Shewanella homologue.
