Chhikara Nidhi, Timilsina Grishma, Wang Yu, Reasons Dexter, Outten F Wayne, Frantom Patrick A
Department of Chemistry and Biochemistry, The University of Alabama, Tuscaloosa, AL 35487, United States.
Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC 29208, United States.
Metallomics. 2025 Aug 5;17(8). doi: 10.1093/mtomcs/mfaf029.
The Suf pathway is the most common pathway for bacterial iron-sulfur cluster assembly and uses the SufBC2D complex as a scaffold for cluster formation. In most Gram-negative bacteria, the SufB subunit of SufBC2D accepts a persulfide from the transpersulfurase, SufE, for incorporation into nascent clusters. There is no reported structure for the SufBC2D-E complex and mechanistic details concerning the coordination of persulfide delivery with other SufBC2D activities are unclear. Using the Suf pathway from Escherichia coli as a model system, we report that SufE acts as a noncompetitive inhibitor of SufBC2D ATPase activity with a Ki value of 1.8 ± 0.2 µM. This value corresponds with a KD value of 1.6 ± 0.2 µM for SufE binding to the SufBC2D complex determined by fluorescence polarization. The rate of persulfide transfer from SufE to SufBC2D is impaired in the presence of ATP, suggesting that the two reactions are mutually exclusive. An AlphaFold3 model of the SufBC2D-E complex predicts electrostatic interactions between acidic residues on SufC and basic residues on the N-terminal helix of SufE. SufE variants at the K9 and R16 positions interfere with the ability of SufE to transfer persulfide to SufBC2D and to inhibit SufBC2D ATPase activity. In vivo complementation growth assays show that these SufE variants exhibit a slow-growth phenotype under iron starvation conditions, confirming the connection between SufE and SufC as important for optimal function in the Suf pathway. The mutual exclusivity of persulfide delivery from SufE and SufBC2D ATPase activity suggests an ordered mechanism for cluster assembly.
Suf途径是细菌铁硫簇组装最常见的途径，它使用SufBC2D复合物作为簇形成的支架。在大多数革兰氏阴性细菌中，SufBC2D的SufB亚基从转硫酶SufE接受一个过硫化物，用于掺入新生簇中。目前尚无SufBC2D-E复合物的报道结构，关于过硫化物传递与SufBC2D其他活性的协调机制细节尚不清楚。以大肠杆菌的Suf途径为模型系统，我们报道SufE作为SufBC2D ATP酶活性的非竞争性抑制剂，Ki值为1.8±0.2µM。该值与通过荧光偏振测定的SufE与SufBC2D复合物结合的KD值1.6±0.2µM相对应。在ATP存在下，过硫化物从SufE转移到SufBC2D的速率受到损害，这表明这两个反应是相互排斥的。SufBC2D-E复合物的AlphaFold3模型预测了SufC上的酸性残基与SufE N端螺旋上的碱性残基之间的静电相互作用。K9和R16位置的SufE变体干扰了SufE将过硫化物转移到SufBC2D并抑制SufBC2D ATP酶活性的能力。体内互补生长试验表明，这些SufE变体在铁饥饿条件下表现出缓慢生长的表型，证实了SufE和SufC之间的联系对Suf途径中的最佳功能很重要。SufE的过硫化物传递与SufBC2D ATP酶活性的相互排斥性表明了一种有序的簇组装机制。
