FeoC from Klebsiella pneumoniae uses its iron sulfur cluster to regulate the GTPase activity of the ferrous iron channel

• Published in: Biochimica et biophysica acta. Proteins and proteomics Vol. 1871; no. 1; p. 140855
• Main Authors: Hsueh, Kuang-Lung, Yu, Liang-Kun, Hsieh, Yin-Cheng, Hsiao, Ya-Yun, Chen, Chun-Jung
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.01.2023
• Subjects: FeoC; Ferrous iron channel; GTP Phosphohydrolases - genetics; GTPase; Iron; Klebsiella pneumoniae; Klebsiella pneumoniae - genetics; NMR; Sodium Chloride; Sulfur; NMR; FeoC; CFeoB; NFeoB; GTPase; Ferrous iron channel; Klebsiella pneumoniae
• ISSN: 1570-9639; 1878-1454
• DOI: 10.1016/j.bbapap.2022.140855

Bacteria depend on the ferrous iron transport (Feo) system for the uptake of ferrous iron (Fe2+). The Feo system is crucial for colonization and virulence of pathogens. In γ-proteobacteria, the system consists of FeoA, FeoB, and FeoC. The function of FeoA remains unknown. FeoB likely forms the channel, whose regulation has been suggested to involve its GTPase domain (part of its NFeoB domain). FeoC from Klebsiella pneumonia was found to contain a [4Fe4S] cofactor, whose presence was speculated to enhance the GTPase activity of FeoB (Hsueh, K.-L., et al., J. Bacteriol. 2013 195(20): 4726–34). We present results here that support and extend that hypothesis. We monitored the GTPase activity of FeoB by NMR spectroscopy and found that the presence of 7% FeoC-[4Fe-4S]3+ (the highest level of cofactor achieved in vitro) increased the GTPase rate of NFeoB by 3.6-fold over NFeoB. The effect depends on the oxidation state of the cluster; with reduction of the cluster to [4Fe-4S]2+ the GTPase greatly decreased the GTPase rate. From the effects of point mutations in FeoC on GTPase rates, we conclude that Lys62 and Lys68 on FeoC each contribute to increased GTPase activity on NFeoB. Mutation of Thr37 of NFeoB to Ser nearly abolished the GTPase activity. The GTPase activity of the isolated K. pneumoniae NFeoB-FeoC complex (NFeoBC) was found to be higher in KCl than in NaCl solution. We solved the X-ray structure of the NFeoBC crystallized from KCl and compared it with a prior X-ray structure crystalized from NaCl. We propose a hypothesis, consistent with these results, to explain the factors that influence the GTPase activity. Bacteria may use the oxygen-sensitive cluster as a sensor to up-regulate the gate closing speed. •We demonstrated that when 7% [4Fe-4S]3+-FeoC (holo-FeoC) is bound to FeoB, the GTPase rate could be enhanced by ∼3.6-fold. This is the first Fe-S protein to regulate G protein.•We tried to perturb the [4Fe-4S]3+ cluster with DTT and found that the DTT-reduced cluster ([4Fe-4S]2+) could no longer activate the GTPase. Also, the key local positive charges (K62 and K68) around the cluster of FeoC were found to be essential to the rate enhancement.•We solved a crystal structure in KCl with missing the secondary structures that support the putative activation mechanism.•Also, we purified a threonine to serine (T37S) mutant FeoB protein and found that the GTPase rate of T37S-FeoB dropped by more than 85%. Last, we summarize the structural evidences to explain the putative activation mechanism.