Structure, Mechanism, and Physiology of Two Sulfur-Mobilizing Enzymes from Synechocystis 6803

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Proteins that contain iron-sulfur (Fe-S) clusters are essential to all organisms. Great progress has been made recently in defining the mechanisms by which these complex clusters are assembled in biology. Components of the so-called Isc Fe-S assembly system are widespread in all three domains of life. The central cog in the Isc machine is a pyridoxal phosphate-dependent cysteine desulfurase, IscS. Multiple accessory proteins interact and cooperate with IscS in Fe-S synthesis/insertion. A distinct system, designated as Suf, has very recently been recognized in bacteria and plants and is likely to be the primary assembly apparatus in plant chloroplasts. A protein (SufS) similar to IscS is likely to be the equivalent sulfur-trafficker for the Suf assembly system. Additional accessory proteins that cooperate with SufS have not yet been identified, and discovery of these factors is one aspect of this research. It has been presumed that the mechanism of the SufS desulfurase is similar to that of the more extensively characterized IscS desulfurase, but preliminary data from the Bollinger lab suggest that there may be important differences. The project will elucidate the mechanism of the SufS reaction and the extent to which it differs from that of IscS. Evidence suggests that the Suf system may be primarily responsible for Fe-S assembly in cyanobacteria. Alternatively, a similar protein (cyst(e)ine C-S lyase or C-DES) that cleaves sulfur from cysteine and its S-substituted derivatives by a distinct mechanism may be the key sulfur-mobilizing Fe-S assembly factor. The project will determine which of these proteins is of primary importance in cyanobacterial Fe-S synthesis, if both are important, or if neither is. Mechanistic characterization of cysteine desulfurases has engendered detailed hypotheses to explain how the desulfurases avoid catalyzing the sulfur elimination reaction promoted by C-DES and, conversely, how C-DES achieves specificity against cysteine in favor of S-substituted derivatives such as cystine and S-(alkyl)cysteines. Predictions based on these hypotheses will be tested in order to evaluate the molecular logic underlying the functional divergence of these similar enzymes. Two areas of chemistry in which Nature's sophistication far exceeds our own are (1) the construction of complex inorganic assemblies for use as the active centers of catalysts and (2) the design of catalysts (enzymes) that promote only one type of reaction among a range of chemically similar pathways that may be available with a given substrate. It is expected that a more profound understanding of Nature's strategies in these two areas would inspire development of new chemical processes. This research seeks a better understanding of Nature's multiple strategies for synthesis of one particularly versatile and important class of complex inorganic assembly, the iron-sulfur clusters. As iron-sulfur clusters are important in biochemical processes ranging from nitrogen fixation to respiration to photosynthesis, it is expected that the results may one day inspire synthetic processes for construction of important biomimetic catalysts. As part of the effort to understand biological iron-sulfur cluster assembly, the project will determine how two similar enzymes that may each have a role in mobilizing the inorganic sulfide needed for the assembly process can select two chemically distinct reaction pathways for breakdown of a common substrate, cysteine. The principles relating the structure of each enzyme to the reaction pathway it promotes will constitute a paradigm for how chemical specificity may be achieved by subtle tuning of catalyst structure.