The Influence of Metal Ions on the Conformation of the SufU Iron‐Sulfur Cluster Biosynthesis Protein from Bacillus subtilis
Authors: Almutairi Hayfa Habes , Zeilstra‐Ryalls Jill Helen , Torelli Andrew Tyler 
Published: June 2021

Iron‐sulfur (Fe‐S) clusters are critical cofactors necessary for the function of myriad proteins, including those involved in respiration, photosynthesis, DNA repair, and more. Fe‐S cluster biosynthesis is nearly universally required in all organisms. Three assembly machineries, ISC, NIF and SUF have been identified, and all are represented among bacteria. Escherichia coli, a Gram− bacterium, has both ISC and SUF systems; most Gram+ bacteria, including Bacillus subtilis and Streptococcus mutans, have only one system (SUF). These Fe‐S assembly systems include a cysteine desulfurase enzyme, either IscS or SufS, which provides the sulfide constituent for Fe‐S cluster assembly. A scaffold protein is another critical component, serving as a platform for cluster assembly and participating in transfer of the clusters to target apoproteins. In E. coli, IscU is recognized as a scaffold protein in the ISC system, whereas SufBCD complex is the scaffold protein in the SUF system. In B. subtilis and S. mutans, SufU has been suggested to be a scaffold protein; however, SufU has also been found to enhance the activity of its cognate SufS desulfurase. Similarities in secondary structure, amino acid sequences, and metal binding to the active sites of IscU and SufU predict that these two U‐type proteins share other properties and functions; specifically, structural dynamics in response to binding metal ions, and also metal ion specificity. This study aims to (1) characterize the behavior of B. subtilis SufU toward two different transition metal ions (Zn2+ and Fe3+), and (2) compare and contrast conformational changes the B. subtilis protein undergoes upon binding these metal ions to those of E. coli IscU and S. mutans SufU. Experimental approaches include colorimetry, Isothermal Titration Calorimetry (ITC), Dynamic Light Scattering (DLS), Circular Dichroism (CD), and tryptophan fluorescence. Colorimetry using 4‐(2‐pyridylazo) resorcinol revealed that SufU was purified with bound Zn2+. Based on ITC, Zn2+ binds to SufU at a single site with Kd value of 7.11 μM. This was corroborated using an assay based on intrinsic tryptophan fluorescence. There was no evidence of Fe3+ binding using either approach. The results indicated that purified SufU that had been stripped of its bound Zn2+ undergoes conformational changes upon reconstitution with Zn2+ ions. By contrast, no indication of such changes was obtained upon the addition of Fe3+ ions. Thus, B. subtilis SufU discriminates between Zn2+ and Fe3+ ions, with preference for the former. These properties are shared with E. coli IscU and S. mutans SufU, suggesting that they are widespread characteristics of U‐type proteins that participate in Fe‐S cluster biosynthesis. The findings for B. subtilis SufU contribute to a broader understanding of the kinds of protein‐protein interactions and structural adaptability that are associated with binding of metal ions to scaffold proteins. They should also be useful in investigating features that are distinctive to subsets of these proteins, such as the Gram Positive Region (GPR), which is absent from the E. coli IscU.

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Bowling Green State University

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.