Protected Sulfur Transfer Reactions by the Escherichia coli Suf System

Abstract

The first step in sulfur mobilization for the biosynthesis of Fe-S clusters under oxidative stress and iron starvation in Escherichia coli involves a cysteine desulfurase SufS. Its catalytic reactivity is dependent on the presence of a sulfur acceptor protein, SufE, which acts as the preferred substrate for this enzyme. Kinetic analysis of the cysteine:SufE sulfurtransferase reaction of the E. coli SufS that is partially protected from reducing agents, such as dithiothreitol and glutathione, was conducted. Under these conditions, the reaction displays a biphasic profile in which the first phase involves a fast sulfur transfer reaction from SufS to SufE. The accumulation of persulfurated/polysulfurated forms of SufE accounts for a second phase of the slow catalytic turnover rate. The presence of the SufBCD complex enhances the activity associated with the second phase, while modestly inhibiting the activity associated with the initial sulfur transfer from SufS to SufE. Thus, the rate of sulfur transfer from SufS to the final proposed SufBCD Fe-S cluster scaffold appears to be dependent on the availability of the final sulfur acceptor. The use of a stronger reducing agent [tris(2-carboxyethyl)phosphine hydrochloride] elicited the maximal activity of the SufS-SufE reaction and surpassed the stimulatory effect of SufBCD. This concerted sulfur trafficking path involving sequential transfer from SufS to SufE to SufBCD guarantees the protection of intermediates at a controlled flux to meet cellular demands encountered under conditions detrimental to thiol chemistry and Fe-S cluster metabolism.

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

“…However, the structure of the E. coli SufS apo -SufE apo complex and potential conformational changes that result from their interaction are not defined. SufE binding increases the SufS desulfurase activity, at least in part, by acting as a co-substrate for the ping-pong reaction pathway that depends on SufE Cys-51 (9,11). In this study, we used HDX-MS, HDX deuterium trapping, and biochemical assays to better define the role protein dynamics plays in possible SufE allosteric activation of SufS catalytic activity.…”

“…It is also known that in the absence of a further sulfur acceptor (e.g. SufB or a thiol reductant), Cys-51 of SufE can accept multiple sulfur groups, thereby forming a polymeric sulfur species (8,11). This indicates that both apo and persulfurated/polysulfurated SufE can bind to SufS, presumably with protrusion of the Cys-51 side chain into the active site cavity, as was observed in the co-structure of CsdA-CsdE in which the CsdE Cys-61 thiolate is exposed and oriented toward Cys-358 of CsdA (31).…”

“…sulfide intermediate form of SufS (SufS per ) stimulates SufS desulfurase activity by providing an acceptor for the persulfide species via direct sulfur transfer (9,11,18). Thus, SufS could have a different conformational response to SufE that is dependent on the SufS catalytic intermediate state.…”

“…The monomeric 15.8-kDa SufE co-substrate protein interacts with the SufS dimer to stimulate cysteine desulfurase activity and accepts sulfane sulfur through a persulfide transfer reaction (8,9). This sulfur transfer reaction, which proceeds via a pingpong mechanism, may be important for limiting sulfide release under oxidative stress conditions (10,11). SufE transfers the persulfide to SufB of the SufBC 2 D complex, which is a scaffold complex that assembles [4Fe-4S] clusters (12)(13)(14).…”

“…In the presence of SufE, SufS cysteine desulfurase activity is increased by an order of magnitude (8,9). The invariant Cys-51 of SufE acts as a co-substrate for SufS and accepts the sulfur from Cys-364 of SufS, thereby enhancing the catalytic rate (9,11,18). It is also possible that interaction with SufE may elicit changes in structural dynamics within the active site that facilitate the desulfuration reaction (5).…”