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

Abstract

The facultative anaerobe Escherichia coli adopts different metabolic modes in response to the availability of oxygen. The global transcriptional regulator FNR (fumarate-nitrate reduction) monitors the availability of oxygen in the environment. Binding as a homodimer to palindromic sequences of DNA, FNR carries a sensory domain, remote from the DNA binding helix-turn-helix motif, which responds to oxygen. 2؉ , causing a similar cluster transformation to a [2Fe-2S] form. Either the hydrogen peroxide formed on reaction with oxygen can be recycled by intracellular catalase or it can be used to oxidize further Fe-S clusters. In both cases, the efficacy of oxygen sensing by FNR will be increased.Escherichia coli is a facultative anaerobe that adopts different metabolic modes in response to the availability of oxygen (1). A hierarchy of metabolism exists in which aerobic respiration is preferred to anaerobic respiration, which in turn is preferred to fermentation (1). In simple terms, the global transcriptional regulator FNR 1 (designated due to defects in fumarate-nitrate reduction of the corresponding mutants), along with other transcription factors, ArcBA, NarXL, NarPQ, and FhlA, maintains this hierarchy by monitoring the availability of oxygen in the environment (1). FNR is a member of a large family of transcription factors that modulate physiological changes in response to a variety of metabolic and environmental challenges (2). Members of the family are predicted to be structurally related to the catabolite gene activator protein, CAP (also known as the cAMP receptor protein). CAP and FNR proteins bind as homodimers to palindromic sequences of DNA, each monomer binding to one half-site (2). They consist of two functionally distinct domains, a DNA binding helix-turn-helix motif and an N-terminal region of antiparallel ␤-strands forming the sensory domain (3). The sensing regions are adapted to respond to different effectors (2). Thus, CAP reversibly binds cAMP to monitor glucose status (4), and the sensing domain of the CooA protein of Rhodospirillum rubrum possesses a b-type cytochrome that binds CO (5). The sensory region of FNR has four conserved cysteine residues 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...

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

“…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). Recently, we reported the detection of approximately two sulfide ions released per FNR monomer during cluster conversion (22), demonstrating that cluster oxidation is metal based.…”

“…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).…”

“…Molecular oxygen triggers the conversion of the [4Fe-4S] 2ϩ cluster into a [2Fe-2S] 2ϩ cluster, both in vivo and in vitro, causing a conformational change within the protein that induces monomerization, preventing sequence-specific DNA binding and favorable interactions with the transcription machinery (12,(14)(15)(16)(18)(19)(20).…”

“…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).…”

“…FNR is activated under anaerobic conditions by the acquisition of one [4Fe-4S] 2ϩ cluster per protein (12)(13)(14)(15), which promotes dimerization and enhances site-specific DNA-binding to target promoters (16,17). Molecular oxygen triggers the conversion of the [4Fe-4S] 2ϩ cluster into a [2Fe-2S] 2ϩ cluster, both in vivo and in vitro, causing a conformational change within the protein that induces monomerization, preventing sequence-specific DNA binding and favorable interactions with the transcription machinery (12,(14)(15)(16)(18)(19)(20).…”