Characterization of [4Fe-4S]-Containing and Cluster-Free Forms of Streptomyces WhiD

Abstract

WhiD, a member of the WhiB-like (Wbl) family of iron-sulfur proteins found exclusively within the actinomycetes, is required for the late stages of sporulation in Streptomyces coelicolor. Like all other Wbl proteins, WhiD has not so far been purified in a soluble form that contains a significant amount of cluster and characterization has relied on cluster-reconstituted protein. Thus, a major goal in Wbl research is to obtain and characterize native protein containing iron-sulfur clusters. Here we report the analysis of S. coelicolor WhiD purified anaerobically from E. coli as a soluble protein containing a single [4Fe-4S] 2+ cluster ligated by four cysteines. Upon exposure to oxygen, spectral features associated with the [4Fe-4S] cluster were lost in a slow reaction that unusually yielded apo-WhiD directly without significant concentrations of cluster intermediates. This process was found to be highly pH dependent with an optimal stability observed between pH 7.0 and 8.0. Low molecular weight thiols, including a mycothiol analogue and thioredoxin, exerted a small but significant protective effect against WhiD cluster loss, an activity that could be of physiological importance.[4Fe-4S] 2+ WhiD was found to react much more rapidly with superoxide than with either oxygen or hydrogen peroxide, which may also be of physiological significance. Loss of the [4Fe-4S] cluster to form apo-protein destabilized the protein fold significantly, but did not lead to complete unfolding. Finally, apo-WhiD exhibited negligible activity in an insulin-based disulfide reductase assay demonstrating that it does not function as a general protein disulfide reductase.WhiB-like (Wbl) proteins are found exclusively in the actinomycetes, a phylum of Grampositive bacteria that includes Streptomyces, the most abundant source of clinically important antibiotics and other bioactive molecules, and medically important pathogens such as Mycobacterium tuberculosis and Corynebacterium diphtheriae (1,2). Disruption of wbl genes has shown that Wbl proteins play critical roles in the biology of both Streptomyces and Mycobacterium (1,3). In Streptomyces coelicolor, WhiB, the founding member of the Wbl 1To whom correspondence should be addressed: A. J. Thomson Table S1; The reactivity of the [4Fe-4S] cluster monitored by CD spectroscopy is shown in Figure S1; The reactivity of the [4Fe-4S] cluster with superoxide is shown in Figure S2; Analysis of apo-and as isolated WhiD by SDS-PAGE is shown in Figure S3; EPR spectroscopy data following oxidation of WhiD is shown in Figure S4. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 December 29. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript family, is required for the initiation of sporulation septation, and WhiD, the subject of this report, is required for the late stages of sporulation (1,3,4). In M. tuberculosis, Wbl proteins have been implicated in...

WhiB7, an Fe-S-dependent Transcription Factor That Activates Species-specific Repertoires of Drug Resistance Determinants in Actinobacteria

“…5A). This spectrum was indistinguishable from anaerobically purified WhiD from S. coelicolor, reported to contain a 4Fe-4S cluster (27). It also lacked the characteristic broad shoulders (424, 460, and 560 -580 nm) of aerobically purified WhiB7 from M. tuberculosis and WhiD from S. coelicolor proteins reported to have 2Fe-2S clusters (19,23).…”

“…Over 95% of the purified protein was WhiB7 ST , as determined by SDS-PAGE analysis (data not shown). The UV-visible spectrum of anaerobically purified WhiB7 ST was similar to anaerobically purified WhiD (27) and reconstituted WhiB proteins containing 4Fe-4S clusters (23). Removal of the N-terminal His tag by enterokinase did not alter the absorbance spectra (data not shown).…”

“…These preparations can be used to reconstitute iron-sulfur clusters; however, proteins assembled in this way contain substoichiometric concentrations of iron (19,22,23,26). To our knowledge, there is only one report describing anaerobic purification of a soluble Wbl protein, presumably in its native form (27). Anaerobically purified WhiD contains a 4Fe-4S cluster having about 4 mol of iron per monomer (Ͼ70% occupancy).…”

Mycobacterium tuberculosisWhiB3: A Novel Iron–Sulfur Cluster Protein That Regulates Redox Homeostasis and Virulence

“…Subsequent reduction of the disulfide bond (through a yet unknown mechanism) stimulates disulfide reductase activity. This debate was fueled further when independent groups were unable to detect such activity with WhiB1 or the S. coelicolor orthologue of WhiB3 (13,42), and also when it was reported that Mtb WhiB1 interacts with GlgB (an a-1,4-glucan branching enzyme) to reduce the intramolecular GlgB disulfide bond (20). Although these enzymatic studies are in contrast with increasing evidence suggesting that the Wbl proteins are regulatory DNA-binding proteins (37,40,42), or transcription factors as was recently demonstrated for Mtb WhiB1 (42), it cannot be discounted that WhiB1 may function as a highly selective reductase.…”

“…In addition, NO can react with the Fe-S clusters of TCA cycle enzymes, leading to the formation of a protein-bound dinitrosyl-iron complex (DNIC), which can alter enzymatic activity (17). Also, in elegant biochemical studies on Streptomyces WhiD (the homologue of Mtb WhiB3) and Mtb WhiB1, protein stability in the presence of H 2 O 2 and O 2 C- (13) and NO (14) What protects the WhiB3 Fe-S cluster against oxidation and eventually complete loss of the cluster during aerobic growth? One possibility is that a mycobacterial redox buffer in the cytoplasm protects cellular Fe-S clusters.…”