Kinetic and Structural Characterization of Slr0077/SufS, the Essential Cysteine Desulfurase from Synechocystis sp. PCC 6803^,

Abstract

Cysteine desulfurases, designated NifS, IscS, and SufS, cleave L-cysteine to form alanine and an enzyme cysteinyl persulfide intermediate. Genetic studies on the photosynthetic cyanobacterium Synechocystis sp. PCC 6803 have shown that of the three Nif/Isc/SufS-like proteins encoded in its genome only the sequence group II protein, Slr0077/SufS, is essential. This protein has been overexpressed in Escherichia coli, purified to homogeneity, shown to bind pyridoxal-5'-phosphate (PLP) and to catalyze cysteine desulfuration, and characterized in terms of its structure and kinetics. The results suggest that catalysis in the absence of accessory factors has two constituent pathways, one involving nucleophilic attack by C372 to form the Slr0077/SufS-bound cysteinyl persulfide intermediate and the second involving intermolecular attack by the sulfur of a second molecule of the substrate on the initial l-cysteine-PLP complex to form free l-cysteine persulfide. The second pathway is operant in the C372A variant protein, explaining why it retains significant activity, which is proportional to the concentration of l-cysteine (i.e., does not saturate). C-S bond cleavage by the first (normal) pathway is considerably less efficient than the equivalent step in a group I desulfurase (Slr0387) from the same organism (characterized in the accompanying paper). The 1.8 A crystal structure of the protein, which is very similar to that previously reported for E. coli SufS, shows that the loop on which C372 resides is well-ordered and shorter by 11 residues than the corresponding disordered loop of the group I NifS-like protein from Thermotoga maritima. Sequence comparisons establish that the T. maritima and Slr0387 proteins have loops of similar length. The combined structural and kinetic data imply that the modest activity of Slr0077/SufS and other SufS proteins in comparison to their sequence group I (NifS/IscS-like) paralogues results from inefficiency in the nucleophilic attack step associated with differences in the structure or dynamics of this loop. The recent reports that SufS proteins can be activated manyfold by binding to SufE thus implies that the accessory protein either accelerates nucleophilic attack by the conserved cysteine residue of SufS by a conformational mechanism or itself contributes a nucleophilic cysteine for more efficient intermolecular attack.

Escherichia coli SufE Sulfur Transfer Protein Modulates the SufS Cysteine Desulfurase through Allosteric Conformational Dynamics

“…Cysteine Binding Assays-All assays were performed at room temperature in Buffer A. L-Cysteine binding was evaluated by monitoring the immediate ⌬A 420 or ⌬A 340 elicited by the addition of increasing concentrations of L-cysteine to 25 M SufS apo or 25 M SufS apo with an equal amount of SufE alk (27). Protein was first added to the cuvettes, and then L-cysteine was added and mixed for ϳ5 s prior to a wavelength scan from 200 to 650 nm.…”

Fe-S Cluster Assembly Pathways in Bacteria

“…A mixture of SufS and SufE, precharged with persulfide, can deposit sulfur on Cys residues in SufA (105). The exact mechanism by which SufE enhances SufS activity is still a matter of debate and could occur by enhancing the initial cleavage of the C-S bond of the cysteine-PLP adduct rather than by accelerating subsequent breakage of the SufS persulfide (118).…”

“…This assignment is supported by the observation that a NifU homologue is colocated with the suf genes in cyanobacteria such as Gloeobacter violaceus. In contrast, the two iscS homologues, iscS1 and iscS2, and the iscA gene are not essential in Synechocystis, so it is unlikely that they are the main sulfur donors for cluster assembly on Nfu (7,11,118). Nfu, encoded by ssl2667, can form a [2Fe-2S] cluster and transfer that cluster in vitro to apoferredoxin (86).…”

“…The relevant im- (7,11,86,118,127). The increased importance of the Suf pathway in Synechocystis likely stems from innate differences between photosynthetic and nonphotosynthetic organisms.…”

Identification of persulfide-binding and disulfide-forming cysteine residues in the NifS-like domain of the molybdenum cofactor sulfurase ABA3 by cysteine-scanning mutagenesis

“…The ubiquitous presence of cysteine206 and corresponding cysteines in other Moco sulfurases and the fact that cysteine206 is exposed on the surface of ABA3-NifS therefore indicates a conserved function of this residue, probably in accepting a persulfide from the active site cysteine430 and its further transfer to the C-terminal domain of ABA3. To verify the hypothesis of a possible persulfide transfer between cysteine430 and cysteine206 as well as the deduced disulfide bond between cysteine428 and cysteine435, a theoretical structure of ABA3-NifS was calculated based on the existing structure of Synechocystis SufS [25]. With an estimated accuracy of approximately 70 %, this structure reveals the position of each cysteine residue in the ABA3-NifS protein (except cysteine456; Fig.…”