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.

The biosynthesis of Fe−S clusters and other thio-cofactors requires the participation of redox agents. A shared feature in these pathways is the formation of transient protein persulfides, which are susceptible to reduction by artificial reducing agents commonly used in reactions in vitro. These agents modulate the reactivity and catalytic efficiency of biosynthetic reactions and, in some cases, skew the enzymes' kinetic behavior, bypassing sulfur acceptors known to be critical for the functionality of these pathways in vivo. Here, we provide kinetic evidence for the selective reactivity of the Bacillus subtilis Trx (thioredoxin) system toward proteinbound persulfide intermediates. Our results demonstrate that the redox flux of the Trx system modulates the rate of sulfide production in cysteine desulfurase assays. Likewise, the activity of the Trx system is dependent on the rate of persulfide formation, suggesting the occurrence of coupled reaction schemes between both enzymatic systems in vitro. Inactivation of TrxA (thioredoxin) or TrxR (thioredoxin reductase) impairs the activity of Fe−S enzymes in B. subtilis, indicating the involvement of the Trx system in Fe−S cluster metabolism. Surprisingly, biochemical characterization of TrxA reveals that this enzyme is able to coordinate Fe−S species, resulting in the loss of its reductase activity. The inactivation of TrxA through the coordination of a labile cluster, combined with its proposed role as a physiological reducing agent in sulfur transfer pathways, suggests a model for redox regulation. These findings provide a potential link between redox regulation and Fe−S metabolism.