Kuang-Lung Hsueh

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.

Simple Summary Grenz rays are low energy X-rays and have been used for dermatological radiotherapy for decades. This study calculates the relative biological effectiveness (RBE) and oxygen enhancement ratio (OER) for DNA double-strand break (DSB) induction and cell survival under hypoxic conditions for Grenz-ray therapy (GT). The RBE values for cell survival for cells irradiated by 15 kV, 10 kV and 10 kVp and titanium K-shell X-rays (4.55 kV) relative to Co-60 gamma-rays are 1.0-1.6 at an aerobic condition (21% O-2) and further increase to 1.2-2.1 in conditions of severe hypoxia (0.1% O-2). The OER values for DSB induction relative to Co-60 gamma-rays are ~2.4 for GT, but the OER for cell survival is 2.8-2.0 as photon energy decreases from 15 kV to 4.55 kV. Grenz-ray therapy (GT) is commonly used for dermatological radiotherapy and has a higher linear energy transfer, relative biological effectiveness (RBE) and oxygen enhancement ratio (OER). GT is a treatment option for lentigo maligna and lentigo maligna melanoma. This study aims to calculate the RBE for DNA double-strand break (DSB) induction and cell survival under hypoxic conditions for GT. The yield of DSBs induced by GT is calculated at the aerobic and hypoxic conditions, using a Monte Carlo damage simulation (MCDS) software. The RBE value for cell survival is calculated using the repair-misrepair-fixation (RMF) model. The RBE values for cell survival for cells irradiated by 15 kV, 10 kV and 10 kVp and titanium K-shell X-rays (4.55 kV) relative to Co-60 gamma-rays are 1.0-1.6 at the aerobic conditions and moderate hypoxia (2% O-2), respectively, but increase to 1.2, 1.4 and 1.9 and 2.1 in conditions of severe hypoxia (0.1% O-2). The OER values for DSB induction relative to Co-60 gamma-rays are about constant and ~2.4 for GT, but the OER for cell survival is 2.8-2.0 as photon energy decreases from 15 kV to 4.55 kV. The results indicate that GT results in more DSB induction and allows effective tumor control for superficial and hypoxic tumors.

IscR is an Fe-S cluster-containing transcription factor involved in a homeostatic mechanism that controls Fe-S cluster biogenesis in Escherichia coli. Although IscR has been proposed to act as a sensor of the cellular demands for Fe-S cluster biogenesis, the mechanism by which IscR performs this function is not known. In this study, we investigated the biochemical properties of the Fe-S cluster of IscR to gain insight into the proposed sensing activity. Mossbauer studies revealed that IscR contains predominantly a reduced [2Fe-2S](+) cluster in vivo. However, upon anaerobic isolation of IscR, some clusters became oxidized to the [2Fe-2S](+) form. Cluster oxidation did not, however, alter the affinity of IscR for its binding site within the iscR promoter in vitro, indicating that the cluster oxidation state is not important for regulation of DNA binding. Furthermore, characterization of anaerobically isolated IscR using resonance Raman, Mossbauer, and nuclear magnetic resonance spectroscopies leads to the proposal that the [2Fe-2S] cluster does not have full cysteinyl ligation. Mutagenesis studies indicate that, in addition to the three previously identified cysteine residues (Cys92, Cys98, and Cys104), the highly conserved His107 residue is essential for cluster ligation. Thus, these data suggest that IscR binds the cluster with an atypical ligation scheme of three cysteines and one histidine, a feature that may be relevant to the proposed function of IscR as a sensor of cellular Fe-S cluster status.