The complex multiprotein systems for the assembly of protein-bound iron sulfur (Fe S) clusters are well defined in Gram-negative model organisms. However, little is known about Fe S cluster biogenesis in other bacterial species. The ISC (iron sulfur cluster) operon of Mycobacterium tuberculosis lacks several genes known to be essential for the function of this system in other organisms. However, the cysteine desulfurase IscSMtb (Rv number Rv3025c; Mtb denotes M. tuberculosis) is conserved in this important pathogen. The present study demonstrates that deleting iscSMtb renders the cells microaerophilic and hypersensitive to oxidative stress. Moreover, the Delta iscSMtb mutant shows impaired Fe S cluster-dependent enzyme activity, clearly indicating that IscSMtb is associated with Fe S cluster assembly. An extensive interaction network of IscSMtb with Fe S proteins was identified, suggesting a novel mechanism of sulfur transfer by direct interaction with apoproteins. Interestingly, the highly homologous IscS of Escherichia coli failed to complement the Delta iscSMtb mutant and showed a less diverse protein-interaction profile. To identify a structural basis for these observations we determined the crystal structure of IscSMtb, which mirrors adaptations made in response to an ISC operon devoid of IscU-like Fe S cluster scaffold proteins. We conclude that in M. tuberculosis IscS has been redesigned during evolution to compensate for the deletion of large parts of the ISC operon.