Jason C. Crack

The transcriptional repressor protein NsrR senses nitric oxide directly via a [2Fe-2S] cluster

The regulatory protein NsrR, a member of the Rrf2 family of transcription repressors, is specifically dedicated to sensing nitric oxide (NO) in a variety of pathogenic and non-pathogenic bacteria. It has been proposed that NO directly modulates NsrR activity by interacting with a predicted [Fe-S] cluster in the NsrR protein, but no experimental evidence has been published to support this hypothesis. Here we report the purification of NsrR from the obligate aerobe Streptomyces coelicolor. We demonstrate using UV-visible, near UV CD and EPR spectroscopy that the protein contains an NO-sensitive [2Fe-2S] cluster when purified from E. coli. Upon exposure of NsrR to NO, the cluster is nitrosylated, which results in the loss of DNA binding activity as detected by bandshift assays. Removal of the [2Fe-2S] cluster to generate apo-NsrR also resulted in loss of DNA binding activity. This is the first demonstration that NsrR contains an NO-sensitive [2Fe-2S] cluster that is required for DNA binding activity.

Bacterial sensors of oxygen.

The concentration of molecular oxygen (O(2)) began to increase in the Earths atmosphere approximately two billion years ago. Its presence posed a threat to anaerobes but also offered opportunities for improved energy conservation via aerobic respiration. The ability to sense environmental O(2) thus became, and remains, important for many bacteria, both for protection and switching between anaerobic and aerobic respiration. Utilizing an iron-sulfur cluster as the sensor of O(2) exploits the ability of O(2) to oxidize the iron-sulfur cluster, ultimately resulting in cluster disassembly. When utilizing heme as the sensor, the capacity of O(2) to form a reversible Fe-O(2) bond or alternatively the oxidation of the heme iron atom itself is used to detect O(2) and switch regulators between active and inactive forms.

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

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. The sensing mechanism involves the transformation of a [4Fe-4S]2+ cluster into a [2Fe-2S] form in vitro on reaction with oxygen. Evidence is presented to show that this process proceeds by at least two steps, the first, an oxidative one, being the formation, on reaction with O2, of a [3Fe-4S]1+ cluster as an intermediate accompanied by the production of hydrogen peroxide. This is followed by a slower, non-redox, pseudo-first order step in which the [3Fe-4S]1+ form converts to a [2Fe-2S]2+ cluster. This must be accompanied by a substantial protein conformational change since the four cysteine ligands that bind the two forms of the FeS clusters have different spatial disposition. Hydrogen peroxide is also an oxidant of the [4Fe-4S]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.

Mechanistic Insight into the Nitrosylation of the [4Fe−4S] Cluster of WhiB-like Proteins

The reactivity of protein bound iron-sulfur clusters with nitric oxide (NO) is well documented, but little is known about the actual mechanism of cluster nitrosylation. Here, we report studies of members of the Wbl family of [4Fe-4S] containing proteins, which play key roles in regulating developmental processes in actinomycetes, including Streptomyces and Mycobacteria, and have been shown to be NO responsive. Streptomyces coelicolor WhiD and Mycobacterium tuberculosis WhiB1 react extremely rapidly with NO in a multiphasic reaction involving, remarkably, 8 NO molecules per [4Fe-4S] cluster. The reaction is 10(4)-fold faster than that observed with O(2) and is by far the most rapid iron-sulfur cluster nitrosylation reaction reported to date. An overall stoichiometry of [Fe(4)S(4)(Cys)(4)](2-) + 8NO → 2[Fe(I)(2)(NO)(4)(Cys)(2)](0) + S(2-) + 3S(0) has been established by determination of the sulfur products and their oxidation states. Kinetic analysis leads to a four-step mechanism that accounts for the observed NO dependence. DFT calculations suggest the possibility that the nitrosylation product is a novel cluster [Fe(I)(4)(NO)(8)(Cys)(4)](0) derived by dimerization of a pair of Roussins red ester (RRE) complexes.

Mycobacterium tuberculosis WhiB1 is an essential DNA-binding protein with a nitric oxide-sensitive iron–sulfur cluster

Mycobacterium tuberculosis is a major pathogen that has the ability to establish, and emerge from, a persistent state. Wbl family proteins are associated with developmental processes in actinomycetes, and M. tuberculosis has seven such proteins. In the present study it is shown that the M. tuberculosis H37Rv whiB1 gene is essential. The WhiB1 protein possesses a [4Fe-4S]2+ cluster that is stable in air but reacts rapidly with eight equivalents of nitric oxide to yield two dinuclear dinitrosyl-iron thiol complexes. The [4Fe-4S] form of WhiB1 did not bind whiB1 promoter DNA, but the reduced and oxidized apo-WhiB1, and nitric oxide-treated holo-WhiB1 did bind to DNA. Mycobacterium smegmatis RNA polymerase induced transcription of whiB1 in vitro; however, in the presence of apo-WhiB1, transcription was severely inhibited, irrespective of the presence or absence of the CRP (cAMP receptor protein) Rv3676, which is known to activate whiB1 expression. Footprinting suggested that autorepression of whiB1 is achieved by apo-WhiB1 binding at a region that overlaps the core promoter elements. A model incorporating regulation of whiB1 expression in response to nitric oxide and cAMP is discussed with implications for sensing two important signals in establishing M. tuberculosis infections.

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

In Escherichia coli, the switch between aerobic and anaerobic metabolism is controlled primarily by FNR (regulator of fumarate and nitrate reduction), the protein that regulates the transcription of >100 genes in response to oxygen. Under oxygen-limiting conditions, FNR binds a [4Fe-4S]2+ cluster, generating a transcriptionally active dimeric form. Upon exposure to oxygen the cluster converts to a [2Fe-2S]2+ form, leading to dissociation of the protein into monomers, which are incapable of binding DNA with high affinity. The mechanism of cluster conversion together with the nature of the products of conversion is of considerable current interest. Here, we demonstrate that [4Fe-4S]2+ to [2Fe-2S]2+ cluster conversion, in both native and reconstituted [4Fe-4S] FNR, proceeds via a one electron oxidation of the cluster, to give a [3Fe-4S]1+ cluster intermediate, with the release of one Fe2+ ion and a superoxide ion. The cluster intermediate subsequently rearranges spontaneously to form the [2Fe-2S]2+ cluster, with the release of a Fe3+ ion and, as previously shown, two sulfide ions. Superoxide ion undergoes dismutation to hydrogen peroxide and oxygen. This mechanism, a one electron activation of the cluster, coupled to catalytic recycling of the resulting superoxide ion back to oxygen, provides a means of amplifying the sensitivity of [4Fe-4S] FNR to its signal molecule.

Reversible cycling between cysteine persulfide-ligated [2Fe-2S] and cysteine-ligated [4Fe-4S] clusters in the FNR regulatory protein

Fumarate and nitrate reduction (FNR) regulatory proteins are O2-sensing bacterial transcription factors that control the switch between aerobic and anaerobic metabolism. Under anaerobic conditions [4Fe-4S]2+-FNR exists as a DNA-binding homodimer. In response to elevated oxygen levels, the [4Fe-4S]2+ cluster undergoes a rapid conversion to a [2Fe-2S]2+ cluster, resulting in a dimer-to-monomer transition and loss of site-specific DNA binding. In this work, resonance Raman and UV-visible absorption/CD spectroscopies and MS were used to characterize the interconversion between [4Fe-4S]2+ and [2Fe-2S]2+ clusters in Escherichia coli FNR. Selective 34S labeling of the bridging sulfides in the [4Fe-4S]2+ cluster-bound form of FNR facilitated identification of resonantly enhanced Cys32S-34S stretching modes in the resonance Raman spectrum of the O2-exposed [2Fe-2S]2+ cluster-bound form of FNR. This result indicates O2-induced oxidation and retention of bridging sulfides in the form of [2Fe-2S]2+ cluster-bound cysteine persulfides. MS also demonstrates that multiple cysteine persulfides are formed on O2 exposure of [4Fe-4S]2+-FNR. The [4Fe-4S]2+ cluster in FNR can also be regenerated from the cysteine persulfide-coordinated [2Fe-2S]2+ cluster by anaerobic incubation with DTT and Fe2+ ion in the absence of exogenous sulfide. Resonance Raman data indicate that this type of cluster conversion involving sulfide oxidation is not unique to FNR, because it also occurs in O2-exposed forms of O2-sensitive [4Fe-4S] clusters in radical S-adenosylmethionine enzymes. The results provide fresh insight into the molecular mechanism of O2 sensing by FNR and iron-sulfur cluster conversion reactions in general, and suggest unique mechanisms for the assembly or repair of biological [4Fe-4S] clusters.

The O2 sensitivity of the transcription factor FNR is controlled by Ser24 modulating the kinetics of [4Fe-4S] to [2Fe-2S] conversion

Fumarate and nitrate reduction regulatory (FNR) proteins are bacterial transcription factors that coordinate the switch between aerobic and anaerobic metabolism. In the absence of O2, FNR binds a [4Fe-4S]2+ cluster (ligated by Cys-20, 23, 29, 122) promoting the formation of a transcriptionally active dimer. In the presence of O2, FNR is converted into a monomeric, non-DNA-binding form containing a [2Fe-2S]2+ cluster. The reaction of the [4Fe-4S]2+ cluster with O2 has been shown to proceed via a 2-step process, an O2-dependent 1-electron oxidation to yield a [3Fe-4S]+ intermediate with release of 1 Fe2+ ion, followed by spontaneous rearrangement to the [2Fe-2S]2+ form with release of 1 Fe3+ and 2 S2− ions. Here, we show that replacement of Ser-24 by Arg, His, Phe, Trp, or Tyr enhances aerobic activity of FNR in vivo. The FNR-S24F protein incorporates a [4Fe-4S]2+ cluster with spectroscopic properties similar to those of FNR. However, the substitution enhances the stability of the [4Fe-4S]2+ cluster in the presence of O2. Kinetic analysis shows that both steps 1 and 2 are slower for FNR-S24F than for FNR. A molecular model suggests that step 1 of the FNR-S24F iron–sulfur cluster reaction with O2 is inhibited by shielding of the iron ligand Cys-23, suggesting that Cys-23 or the cluster iron bound to it is a primary site of O2 interaction. These data lead to a simple model of the FNR switch with physiological implications for the ability of FNR proteins to operate over different ranges of in vivo O2 concentrations.

Iron–sulfur cluster sensor-regulators

Regulatory proteins that contain an iron-sulfur cluster cofactor constitute a group that is growing both in number and importance, with a range of functions that include sensing of molecular oxygen, stress response, and iron regulation. In all cases, the cluster plays a central role, as a sensory module, in controlling the activity of the regulator. In some cases, the cluster is required for the protein to attain its regulatory form, while in others the active form requires loss or modification of the cluster. In this way, nature has exploited the inherent reactivity of iron-sulfur clusters. Here, we focus on recent advances that provide new insight into the remarkable chemistries exhibited by these regulators, and how they achieve the required levels of sensitivity and specificity.

Bacterial iron-sulfur regulatory proteins as biological sensor-switches

SIGNIFICANCE In recent years, bacterial iron-sulfur cluster proteins that function as regulators of gene transcription have emerged as a major new group. In all cases, the cluster acts as a sensor of the environment and enables the organism to adapt to the prevailing conditions. This can range from mounting a response to oxidative or nitrosative stress to switching between anaerobic and aerobic respiratory pathways. The sensitivity of these ancient cofactors to small molecule reactive oxygen and nitrogen species, in particular, makes them ideally suited to function as sensors. RECENT ADVANCES An important challenge is to obtain mechanistic and structural information about how these regulators function and, in particular, how the chemistry occurring at the cluster drives the subsequent regulatory response. For several regulators, including FNR, SoxR, NsrR, IscR, and Wbl proteins, major advances in understanding have been gained recently and these are reviewed here. CRITICAL ISSUES A common theme emerging from these studies is that the sensitivity and specificity of the cluster of each regulatory protein must be exquisitely controlled by the protein environment of the cluster. FUTURE DIRECTIONS A major future challenge is to determine, for a range of regulators, the key factors for achieving control of sensitivity/specificity. Such information will lead, eventually, to a system understanding of stress response, which often involves more than one regulator.