The FeoC [4Fe–4S] Cluster Is Redox-Active and Rapidly Oxygen-Sensitive

Abstract

The acquisition of iron is essential to establishing virulence among most pathogens. Under acidic and/or anaerobic conditions, most bacteria utilize the widely-distributed ferrous iron (Fe2+) uptake (Feo) system to import metabolically-required iron. The Feo system is inadequately understood at the atomic, molecular, and mechanistic levels, but we do know it is composed of a main membrane component (FeoB) essential for iron translocation, as well as two small, cytosolic proteins (FeoA and FeoC) hypothesized to function as accessories to this process. FeoC has many hypothetical functions, including that of an iron-responsive transcriptional regulator. Here, we demonstrate for the first time that Escherichia coli FeoC (EcFeoC) binds an [Fe-S] cluster. Using electronic absorption, X-ray absorption, and electron paramagnetic resonance spectroscopies, we extensively characterize the nature of this cluster. Under strictly anaerobic conditions after chemical reconstitution, we demonstrate that EcFeoC binds a redox-active [4Fe-4S]2+/+ cluster that is rapidly oxygen-sensitive and decays to a [2Fe-2S]2+ cluster (t½ ≈ 20 s), similar to the [Fe-S] cluster in the fumarate and nitrate reductase (FNR) transcriptional regulator. We further show that this behavior is nearly identical to the homologous K. pneumoniae FeoC, suggesting a redox-active, oxygen-sensitive [4Fe-4S]2+ cofactor is a general phenomenon of cluster-binding FeoCs. Finally, in contrast to FNR, we show that [4Fe-4S]2+ cluster binding to FeoC is associated with modest conformational changes of the polypeptide, but not protein dimerization. We thus posit a working hypothesis in which the cluster-binding FeoCs may function as oxygen-sensitive iron sensors that fine-tune pathogenic ferrous iron acquisition.

Iron-sulfur clusters are involved in post-translational arginylation

“…405 nm, common for [4Fe-4S] 2+ clusters. [47][48][49] We further confirmed this assignment using XAS and paramagnetic NMR spectroscopies. XAS again showed a first inflection point of the edge at ≈ 7120 eV and an intense In the absence of sodium dithionite, an axial EPR spectrum is present that reveals the presence of some degraded [3Fe-4S] + cluster (gray), while a [4Fe-4S] 2+ cluster is clearly observed in the dithionite-reduced EPR spectrum (black) of anoxically purified ScATE1.…”

“…3e). 47,56,57 Thus, when our anoxic reconstitution and purification results are taken together, it becomes evident that ScATE1 binds a higher-order cluster in the absence of oxygen and in vivo.…”

“…405 nm, which closely resembles a mixture of [3Fe-4S] and [4Fe-4S] clusters (Supplementary Figure 5). [47][48][49]53,54 We surmise that this mixture is the result of partial or incomplete cluster transfer to ScATE1 during anaerobic cell cultivation and heterologous protein production, or due to extreme O2 sensitivity in the presence of a minute amount of residual O2 during anoxic purification. To confirm these assignments, we collected the CW X-band EPR spectra of ScATE1 expressed and purified under anoxic conditions, both with and without the presence of sodium dithionite.…”

“…Instead, we generated a construct of N-terminal ScATE1 fused to maltose-binding protein (MBP ScATE1-N; Supplementary Figure 8a), an effective strategy in previous methods for recombinant production of small proteins/domains that bind [Fe-S] clusters. 47 We then purified this fusion protein (Supplementary Figure 8b) and subjected it to chemical reconstitution under anoxic conditions.…”

“…475 nm (shoulder), and a broad absorbance envelope from 500 nm to 700 nm, similar to proteins bearing [2Fe-2S] 2+ ferredoxin-like clusters. [43][44][45][46][47] This absorbance spectrum is accompanied by a weak circular dichroism (CD) spectrum in the visible region (Supplementary Figure 2), indicating the presence of the cluster within the polypeptide.…”