Iron-sulfur cluster disassembly in the FNR protein of Escherichia coli by O ₂ : [4Fe-4S] to [2Fe-2S] conversion with loss of biological activity

Abstract

The transcription factor FNR (fumarate nitrate reduction) requires the presence of an iron-sulfur (Fe-S) cluster for its function as a global transcription regulator in Escherichia coli when oxygen becomes scarce. The ability to adapt to changes in oxygen concentrations in the environment is common to many organisms. In the facultative anaerobe, Escherichia coli, the transcription factor FNR ( fumarate nitrate reduction) regulates a network of genes that facilitates adaptation to oxygen deprivation by providing alternative pathways for energy generation (1). Recent data suggest that FNR contains a [4Fe-4S] cluster (2-4) and that this cluster apparently mediates the sensitivity of this transcription factor to oxygen, thus limiting FNR activity to anaerobic conditions. The stoichiometry of iron and labile sulfide relative to the number of cysteine ligands of anaerobically purified FNR is most compatible with the presence of a [4Fe-4S] cluster (3). Iron and sulfide analyses and CD spectra of FNR preparations derived from reconstitution of a cluster into apoprotein also supports this cluster assignment (4). The presence of the Fe-S cluster in the anaerobically purified form of FNR is correlated with an increase in dimerization and specific DNA binding (3), compared with the aerobically purified form that lacks an Fe-S cluster (2, 5, 6). Furthermore, the Fe-S cluster is disrupted by oxygen, and this is correlated with the conversion of FNR into an inactive monomeric protein (3). The loss of this cluster as well as the loss of specific DNA binding upon exposure of FNR to oxygen suggested that the [4Fe-4S] cluster, through its intrinsic instability, serves as an oxygen sensor.This paper reports observations on the path of disassembly of the [4Fe-4S] cluster of FNR in vitro. Little is known about the cluster disassembly mechanism of proteins in the absence of chelators, detergents, chemical oxidants, and other nonphysiological agents. The rapid destruction of the Fe-S cluster of FNR, simply upon exposure of a purified protein solution to air, offers an opportunity to obtain information on the progress and mode of the disassembly of the [4Fe-4S] cluster of this protein. We have used chemical analysis for iron and sulfide as well as electronic, EPR, and Mössbauer spectroscopies to monitor the disassembly process. We report here the unexpected observation that the [4Fe-4S] cluster of FNR is converted in less than 5 min to a [2Fe-2S] cluster in about 60% yield as judged from Mössbauer spectra and this conversion results in a reduction in DNA-binding ability. The [4Fe-4S] 2ϩ cluster can be regenerated from the [2Fe-2S] cluster or its components by reduction with dithionite.

The transcription factor DNR from Pseudomonas aeruginosa specifically requires nitric oxide and haem for the activation of a target promoter in Escherichia coli

“…The mechanism whereby DNR can sense the signal molecule is not yet understood; it is certain, however, that DNR, which lacks conserved cysteines, is unable to form an Fe-S centre that can interact with the signal molecule as reported for the O 2 sensor FNR (Khoroshilova et al, 1995(Khoroshilova et al, , 1997 Green et al, 1996;Jordan et al, 1997 addition, the only evidence in the literature in which a member of the DNR class of regulators binds the target DNA in vitro was obtained by an electophoretic mobility shift assay using the P. aeruginosa DNR protein bound to haem (Giardina et al, 2008). The hypothesis of a haembased sensing is reasonable also in the light of a similar haem-based mechanism well known for the Rhodospirillum rubrum CooA protein (Shelver et al, 1997); in this case the CO molecule binds to the haem iron and triggers a conformational change, thus regulating the transcriptional activity (Lanzilotta et al, 2000).…”

“…The mechanism whereby DNR can sense the signal molecule is not yet understood; it is certain, however, that DNR, which lacks conserved cysteines, is unable to form an Fe-S centre that can interact with the signal molecule as reported for the O 2 sensor FNR (Khoroshilova et al, 1995(Khoroshilova et al, , 1997Green et al, 1996;Jordan et al, 1997). Moreover, spectroscopic evidence supports the hypothesis that haem is involved in NO sensing; in fact the DNR-haem complex is stable and can bind NO in the ferrous state to yield a pentacoordinate derivative (Giardina et al, 2008).…”

Superoxide-mediated amplification of the oxygen-induced switch from [4Fe-4S] to [2Fe-2S] clusters in the transcriptional regulator FNR

“…The C-terminal DNA-binding domain recognizes specific FNRbinding sequences within FNR-controlled promoters (10). The N-terminal sensory domain contains five cysteine residues, four of which (12,(14)(15)(16)(18)(19)(20).The mechanism of the oxygen-mediated cluster conversion is of considerable current interest. Various mechanisms have been proposed for this process, including oxygen reduction to hydrogen peroxide through metal-centered oxidation (15) and oxygen reduction to water through sulfide-based oxidation (21).…”

“…The C-terminal DNA-binding domain recognizes specific FNRbinding sequences within FNR-controlled promoters (10). The N-terminal sensory domain contains five cysteine residues, four of which (12,(14)(15)(16)(18)(19)(20).…”

Mechanism of Oxygen Sensing by the Bacterial Transcription Factor Fumarate-Nitrate Reduction (FNR)

“…The sensory region of FNR has four conserved cysteine residues (Cys-20, -23, -29, and -122) that are essential for in vivo activity and capable of ligating an oxygen-sensitive [4Fe-4S] iron-sulfur cluster (6 -11). A variety of studies have revealed that the active form of FNR contains one [4Fe-4S] 2ϩ cluster/protein monomer that is converted to a [2Fe-2S] 2ϩ cluster, together with other, less well defined iron species, following exposure to oxygen both in vitro and in vivo (10,(12)(13)(14)(15). The switch from a cubane [4Fe-4S] cluster, bound to the protein by four cysteine thiol ligands that sit at the vertices of a tetrahedron, to a planar [2Fe-2S] cluster, also thought to possess four cysteine ligands but lying in a plane, suggests that cluster transformation on exposure to oxygen will provide a large conformational change in the N-terminal region of FNR, presumably thereby initiating the switch of the protein from a DNA binding state to one incapable of binding DNA (8).…”