Antioxidants & Redox Signaling

Iron–sulfur cluster proteins carry out multiple functions, including as regulators of gene transcription/translation in response to environmental stimuli. In all known cases, the cluster acts as the sensory module, where the inherent reactivity/fragility of iron–sulfur clusters with small/redox-active molecules is exploited to effect conformational changes that modulate binding to DNA regulatory sequences. This promotes an often substantial reprogramming of the cellular proteome that enables the organism or cell to adapt to, or counteract, its changing circumstances.

Significant progress has been made recently in the structural and mechanistic characterization of iron–sulfur cluster regulators and, in particular, the O₂ and NO sensor FNR, the NO sensor NsrR, and WhiB-like proteins of Actinobacteria. These are the main focus of this review.

Striking examples of how the local environment controls the cluster sensitivity and reactivity are now emerging, but the basis for this is not yet fully understood for any regulatory family.

Characterization of iron–sulfur cluster regulators has long been hampered by a lack of high-resolution structural data. Although this still presents a major future challenge, recent advances now provide a firm foundation for detailed understanding of how a signal is transduced to effect gene regulation. This requires the identification of often unstable intermediate species, which are difficult to detect and may be hard to distinguish using traditional techniques. Novel approaches will be required to solve these problems.

The Escherichia coli regulatory protein fumarate nitrate reduction (FNR) mediates a global transcriptional response upon O₂ deprivation. Spanning nearly 40 years of research investigations, our understanding of how FNR senses and responds to O₂ has considerably progressed despite a lack of structural information for most of that period. This knowledge has established the paradigm for how facultative anaerobic bacteria sense changes in O₂ tension.

Recently, the X-ray crystal structure of Aliivibrio fischeri FNR with its [4Fe-4S] cluster cofactor was solved and has provided valuable new insight into FNR structure and function. These findings have alluded to the conformational changes that may occur to alter FNR activity in response to O₂.

Here, we review the major features of this structure in context of previously acquired data. In doing so, we discuss additional mechanistic aspects of FNR function that warrant further investigation.

To complement the [4Fe-4S]-FNR structure, the structures of apo-FNR and FNR bound to DNA or RNA polymerase are needed. Together, these structures would elevate our understanding of how ligation of its [4Fe-4S] cluster allows FNR to regulate transcription according to the level of environmental O₂.

Heme binds to and serves as a cofactor for a myriad of proteins that are involved in diverse biological processes. Hemoproteins also exhibit varying modes of heme binding, suggesting that the protein environment contributes to the functional versatility of this prosthetic group. The subject of this review is a subset of hemoproteins that contain at least one heme regulatory motif (HRM), which is a short sequence containing a Cys-Pro core that, in many cases, binds heme with the Cys acting as an axial ligand.

As more details about HRM-containing proteins are uncovered, some underlying commonalities are emerging, including a role in regulating protein stability. Further, the cysteines of some HRMs have been shown to form disulfide bonds. Because the cysteines must be in the reduced, dithiol form to act as a heme axial ligand, heme binds at these sites in a redox-regulated manner, as demonstrated for heme oxygenase-2 (HO2) and Rev-erbβ.

HRM-containing proteins have wide variations in heme affinity, utilize different axial ligand schemes, and exhibit differences in the ability to act as a redox sensor—all while having a wide variety of biological functions. Here, we highlight HO2 and Rev-erbβ to illustrate the similarities and differences between two hemoproteins that contain HRMs acting as redox sensors.

HRMs acting as redox sensors may be applicable to other HRM-containing proteins as many contain multiple HRMs and/or other cysteine residues, which may become more evident as the functional significance of HRMs is probed in additional proteins.

Iron is required for growth and is often redox active under cytosolic conditions. As a result of its facile redox chemistry, iron homeostasis is intricately involved with oxidative stress. Bacterial adaptation to iron limitation and oxidative stress often involves ferric uptake regulator (Fur) proteins: a diverse set of divalent cation-dependent, DNA-binding proteins that vary widely in both metal selectivity and sensitivity to metal-catalyzed oxidation.

Bacteria contain two Fur family metalloregulators that use ferrous iron (Fe²⁺) as their cofactor, Fur and PerR. Fur functions to regulate iron homeostasis in response to changes in intracellular levels of Fe²⁺. PerR also binds Fe²⁺, which enables metal-catalyzed protein oxidation as a mechanism for sensing hydrogen peroxide (H₂O₂).

To effectively regulate iron homeostasis, Fur has an Fe²⁺ affinity tuned to monitor the labile iron pool of the cell and may be under selective pressure to minimize iron oxidation, which would otherwise lead to an inappropriate increase in iron uptake under oxidative stress conditions. Conversely, Fe²⁺ is bound more tightly to PerR but exhibits high H₂O₂ reactivity, which enables a rapid induction of peroxide stress genes.

The features that determine the disparate reactivity of these proteins with oxidants are still poorly understood. A controlled, comparative analysis of the affinities of Fur/PerR proteins for their metal cofactors and their rate of reactivity with H₂O₂, combined with structure/function analyses, will be needed to define the molecular mechanisms that have facilitated this divergence of function between these two paralogous regulators.

The molecule nitric oxide (NO) has been shown to regulate behaviors in bacteria, including biofilm formation. NO detection and signaling in bacteria is typically mediated by hemoproteins such as the bis-(3′,5′)-cyclic dimeric adenosine monophosphate-specific phosphodiesterase YybT, the transcriptional regulator dissimilative nitrate respiration regulator, or heme-NO/oxygen binding (H-NOX) domains. H-NOX domains are well-characterized primary NO sensors that are capable of detecting nanomolar NO and influencing downstream signal transduction in many bacterial species. However, many bacteria, including the human pathogen Pseudomonas aeruginosa, respond to nanomolar concentrations of NO but do not contain an annotated H-NOX domain, indicating the existence of an additional nanomolar NO-sensing protein (NosP).

A newly discovered bacterial hemoprotein called NosP may also act as a primary NO sensor in bacteria, in addition to, or in place of, H-NOX. NosP was first described as a regulator of a histidine kinase signal transduction pathway that is involved in biofilm formation in P. aeruginosa.

The molecular details of NO signaling in bacteria are still poorly understood. There are still many bacteria that are NO responsive but do encode either H-NOX or NosP domains in their genomes. Even among bacteria that encode H-NOX or NosP, many questions remain.

The molecular mechanisms of NO regulation in many bacteria remain to be established. Future studies are required to gain knowledge about the mechanism of NosP signaling. Advancements on structural and molecular understanding of heme-based sensors in bacteria could lead to strategies to alleviate or control bacterial biofilm formation or persistent biofilm-related infections.