Introduction
After the development of oxygenic photosynthesis, the early atmosphere of the Earth was dramatically altered by the presence of the photosynthetic byproduct, oxygen. Organisms evolved a variety of systems to exploit the benefits of atmospheric oxygen while limiting oxygen toxicity. To regulate genes in response to oxygen, some key transcription factors contain oxygen-sensitive [Fe-S] cluster cofactors that act as sensors of oxygen or reactive oxygen species1. Recent work by Crack et al. has provided additional insight into the mechanism of [Fe-S] cluster oxidation and chemical conversion that controls one such transcription factor: the global oxygen-responsive regulator FNR in E. coli2.
[Fe-S] centers function as enzyme cofactors to conduct a variety of electron transfer and substrate activation reactions. However, some [Fe-S] clusters are unstable in the presence of molecular oxygen3. For instance, [4Fe-4S]2+ clusters in the dehydratase family of enzymes are highly sensitive to oxidation and are damaged by oxygen and reactive oxygen species4, 5. Damage occurs when reactive oxygen species oxidize an exposed Fe2+ atom in the cluster. This metal oxidation reaction raises the formal oxidation state of the cluster to [4Fe-4S]3+, which is unstable and degrades. Cluster degradation results in ejection of an iron atom from the cluster and subsequent reduction of the cluster to the inactive [3Fe-4S]+ oxidation state.
FNR (regulator of fumarate and nitrate reduction) regulates the transcription of a large array of genes in response to changes in oxygen levels6. Under anaerobic conditions, FNR binds DNA as a dimer. However, the FNR dimer can only form between FNR monomers that have a [4Fe-4S]2+ cluster. As oxygen levels rise, the oxygen-labile [4Fe-4S]2+ cluster is oxidized and converted to a [2Fe-2S]2+ cluster. FNR cluster conversion is sensitive and rapid, occurring on a scale of minutes after exposure to oxygen7. Conversion of the [4Fe-4S]2+ cluster to a [2Fe-2S]2+ cluster presumably alters FNR's conformation as the cluster binding site shifts to accommodate a planar [2Fe-2S] cluster rather than the cubane [4Fe-4S] cluster. The [2Fe-2S]2+ FNR monomer cannot form the active FNR dimer. Thus cluster conversion acts as a switch to control the DNA binding activity of FNR. As a number of key proteins use sensitive [4Fe-4S] clusters as regulatory switches (see below), defining the mechanism of FNR cluster oxidation should have broad implications for oxygen-responsive gene regulation.
Crack et al.2 have carefully elucidated the mechanism of the FNR [4Fe-4S]2+ cluster oxidation; the mechanism demonstrates that the FNR transcription factor has exploited some aspects of the deleterious oxidation of [Fe-S] clusters to serve a positive role in gene regulation (Fig. 1a). Their work suggests that FNR cluster conversion is a multistep process involving a fast oxidation of the cluster to the [4Fe-4S]3+ form that rapidly decomposes to [3Fe-4S]+, releasing one Fe2+ in the process. The [3Fe-4S]+ cluster intermediate is itself not stable and may directly convert to the [2Fe-2S]2+ form, releasing Fe3+ and two S2- species2, 8, although the details of this second step await further study. The oxygen that oxidizes FNR is in turn reduced, producing both superoxide and hydrogen peroxide as products of the reaction. The studies by Crack et al.2 show that the FNR [4Fe-4S]2+ cluster is poised to be oxidized, resulting in a thermodynamically labile [3Fe-4S]+ form that could then convert to the [2Fe-2S]2+ state. In addition, the observed superoxide and hydrogen peroxide products can be dismutated in vivo by superoxide dismutase and catalase to partially regenerate the 'consumed' oxygen, allowing one oxygen molecule to potentially oxidize up to four [4Fe-4S] clusters (Fig. 1b). Alternatively, hydrogen peroxide itself has been shown to oxidize the FNR cluster and may amplify the FNR response to oxygen through that mode. Superoxide can catalyze the further destruction of the [2Fe-2S]2+ cluster, thus forming apo-FNR, which has been shown to predominate in vivo under fully aerobic conditions9. These results provide a possible biochemical mechanism for FNR activation and shed light on other regulatory systems controlled by [Fe-S] sensors.
Figure 1: Triggering the [Fe-S] switch to regulate FNR.
![Figure 1 : Triggering the [Fe-S] switch to regulate FNR.](/nchembio/journal/v3/n4/thumbs/nchembio0407-206-F1.gif)
(a) Proposed mechanism of FNR cluster oxidation and conversion. (b) Summary scheme of proposed reaction for FNR cluster oxidation by oxygen. Dismutation of superoxide and hydrogen peroxide by superoxide dismutase and catalase in vivo is assumed in the equation.
Full size image (23 KB)An [Fe-S] sensor switch is also used by iron regulatory protein (IRP1) in humans, in which the assembly and disassembly of a [4Fe-4S] cluster controls IRP1 binding to target mRNAs10. [Fe-S] sensors can also be found in some enzymes, such as glutamine 5-phosphoribosyl-1-pyrophosphate (PRPP) amidotransferase from Bacillus subtilis, which has an oxygen-labile [4Fe-4S] cluster that is thought to regulate protein stability and sensitivity to degradation11. The next step to broaden our understanding of the function of these [Fe-S] sensors is to determine how the protein environment controls the reactivity of [Fe-S] clusters. Clearly other amino acids, in addition to the cysteine ligands, must influence the reactivity of the cluster toward oxygen by altering the cluster redox potential or by controlling oxidant access to the cluster. Indeed, specific [Fe-S] clusters may be modulated to sense only a subset of reactive oxygen species. For instance, the [2Fe-2S] cluster present in the SoxR transcription factor responds to superoxide and nitric oxide but is not activated by hydrogen peroxide in vivo1. The protein environment may also enhance or diminish the stability of intermediate cluster forms, such as the [3Fe-4S]+ cluster seen after FNR oxidation, thereby poising the cluster for certain types of conversions. These questions may be partially answered by accurate measurements of the redox potentials of the clusters, as well as high-resolution three-dimensional structures of the [Fe-S] cluster binding sites within the regulatory proteins. This study's detailed exploration of [Fe-S] cluster oxidation and conversion has provided a foundation for addressing these complex possibilities.
