B acterial R edox S ensors

1. Bacterial Redox Sensors Chao Wang Oct 5, 2005

2. Jeffrey Green and Mark S. PagetBacterial redox sensorsNat Rev Microbiol. 2004 Dec;2(12):954-66. Review.Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, University of Sheffield, United Kingdom.

3. Redox reactions pervade living cells.The ability to maintain redox balance is therefore vital to all organisms.Various regulatory sensors continually monitor the redox state of the internal and external environments and control the processes that work to maintain redox homeostasis.These sensors convert the redox signals into regulatoryoutputs, usually at the level of transcription,which allowsthe bacterium to adapt to the altered redox environment.

4. Some well-characterized bacterialredox sensors andtheir mechanismsrelate to biological functions • Thiol-based redox sensors • Fe–S cluster-based sensors • Haem-based sensors • Flavin cofactor-based redox sensors • Pyridine nucleotides • Quinone redox sensors

5. Thiol-based redox sensors • Thiol-based sensor function is reviewed.Typically, these sensors use cysteine modification to sense redox alterations. Examples include OxyR in Escherichia coli, the R-RsrA system in Streptomyces coelicolor, CrtJ and the RegB−RegA in Rhodobacter sphaeroides, and OhrR from Bacillus subtilis. • Cysteine is uniquelysuited to sensing a range of redox signals because thethiol side-chain can be oxidized to several different redoxstates, many of which are readily reversible

6. OxyR — a sensor of peroxide stress • Despite there beinglittle doubt that OxyR uses one or more of its six cysteinethiols to directly sense oxidative stress, the precisenature of the thiol modifications and their consequencesfor OxyR activity are the subject of ongoing controversy. • Storz laboratory supported the role of just one,Cys199, in the hydrogen peroxide-mediated activationof OxyR, possibly through the formation of sulphenicacid (SOH). Stamler laboratoryshowed that OxyR could also be activated by S-nitrosylationwhen treated with S-nitrosothiols (SNOs), therebysupporting the role of the same single cysteine in OxyRactivation. • Storz suggesteda role for another cysteines,Cys208, following the detection in oxidized OxyR of anintramolecular disulphide bond between Cys199 andCys208. • Stamler’s group recently came to theconclusion that Cys208 might not have a role in OxyRactivation15. Instead, they presented evidence thatOxyR is modified by peroxide stress to Cys199-SOH;by nitrosative stress to Cys199-SNO; or by disulphidestress to form a mixed disulphide with glutathione(Cys199-S-SG)

8. σR-RsrA — a sensor of disulphide stress

9. Thiol-based regulators ofanoxygenic photosynthesis • The facultatively photosynthetic bacterium Rhodobactercapsulatus can derive energy from photosynthesis onlywhen the oxygen tension falls below ~2.5%. Expression of the photosystem genes is controlled bymultiple regulators, two of which, CrtJ and theRegB–RegA TWO-COMPONENT SYSTEM, seem to invokereversible disulphide-bond formation as part of theirredox-sensing mechanism. • CrtJaerobic conditions contain two central PAS(PER–ARNT–SIM) domains — a motif that isimplicated in sensing many environmental signals. • RegB–RegA systemanaerobicconditions the inhibition of RegB autophosphorylation underoxidizing conditions correlates with its accumulationin a tetrameric form, and that an intermoleculardisulphide bond involving a single cysteine, Cys265,mediates this oligomerization.Mutation of Cys265 toserine led to increased photosystem gene expressionduring aerobiosis. • How a ‘stable’ disulphide can form in the reducingenvironment of the cytoplasm.

10. OhrR — a sensor modulated by sulphenic acid formation A single redox-sensing cysteine (Cys15) in the Bacillus subtilis OhrR provides perhaps the best evidence for the role of a stable sulphenic acid in modulating regulator activity. Biochemical studies indicate that Cys15 of OhrR is rapidly oxidized to Cys15-SOH when treated with cumene hydroperoxide. This oxidation inhibits DNA-binding, thereby inducing expression of the organic hydroperoxidase gene ohr.

11. Fe–S cluster-based sensors • Fe–S proteinshaveimportant roles as redox-responsive transcriptional andpost-transcriptional regulators in many bacteria. • The importance of Fe−S proteins in redox sensing is illustrated by the functions of several redox sensors from E. coli that use oxidation of Fe−S clusters to monitor the redox status of cell compartments and the environment to produce appropriate transcriptional responses — these include SoxR, Fnr, aconitase and IcsR.

12. SoxR — a sensor of superoxide and nitric-oxide stress • The E. coli SoxR protein is a homodimer that containsone [2Fe–2S] cluster per subunit. • When cultures of E. coli are exposed toconditions that promote the generation of superoxide(for example, in the presence of paraquat and oxygen),the [2Fe–2S]1+ clusters are oxidized to [2Fe–2S]2+ clusters.Oxidation facilitates SoxR-dependent distortion of thesoxS promoter DNA to form a transcriptionally activecomplex with RNA polymerase. Once the source ofthe oxidative stress is removed, the SoxR Fe–S clustersare rapidly reduced, thereby switching off expressionof the SoxRS regulon.

14. Fnr — a sensor of environmental oxygen • Facultative anaerobes such as E. coli adopt differentmetabolic modes in response to oxygen availability.There is a hierarchy of metabolism in which aerobicrespiration (in which oxygen is the terminal electronacceptor) is preferred over anaerobic respiration (inwhich alternative electron acceptors, such as nitrate, areused), which in turn is preferred over fermentation. • Fnr consists of two domains, a carboxyterminalDNA-binding region, which recognizes aspecific DNA sequence in target promoters, and anamino-terminal sensory domain that contains fouressential cysteine residues capable of binding either a[4Fe–4S]2+ or a [2Fe–2S]2+ cluster.

15. During aerobic growth it is suggestedthat Fnr cycles between the active [4Fe–4S]2+ homodimericform and the inactive monomeric apo-protein. Assuming that the rate of cluster synthesis is slower than that of oxygen-dependent cluster degradation, Fnr will mostly be present in the inactive apo-state under aerobic conditions.Anaerobic conditions block the cycle at the [4Fe–4S]2+ to [2Fe–2S]2+ conversion step, thereby allowing the active Fnr dimer [4Fe–4S]2+ to accumulate and anaerobic gene expression to be switched on.

17. Haem-based sensors • The haem is most often bound to a PAS (PER−ARNT−SIM) domainbut can reside within other protein folds. Often, the state of haem is coupled to a transmitterdomain, which transduces the signal into anappropriate output.

18. Dos — an oxygen and redoxsensor • It has an N-terminal haem-bindingPAS domain. However, the sensory domain islinked to a C-terminal phosphodiesterasedomain, rather than a histidine kinase domain,which degrades cyclic AMP in a redox-dependentmanner. • Dos is a tetramer inwhich each subunit has two PAS domains — PAS-A and and PAS-B. Thesteady-state haem configuration in Dos is a six coordinate,with Met95 acting as a second axialligand. • When oxygenbinds to the haem,Met95 is displaced, promotingconformational changes in the PAS-A domain thatinhibit phosphodiesterase activity. However,phophodiesterase activity has also been shown to beregulated by the redox state of the haem iron, leadingto the proposal that Dos is a redox sensor rather thanan oxygen sensor.

19. either oxygen bindingtoferrous haem, or oxidation of the haem moiety, initiatesthe conformational changes that are needed toinhibit phosphodiesterase activity.

20. Flavin cofactor-based redox sensors • The flavin cofactors FAD (flavin adenine dinucleotide) and FMN (flavin mononucleotide)are versatile, electron-carrying coenzymesinvolved in both one- and two-electron transfers. Thecoenzymes can exist in three states: fully oxidized (forexample, FAD); partially oxidized, as a semiquinoneradical (for example, FADH•); and fully reduced (forexample, FADH2). In the past few years, severalFAD-containing primary redox sensors have beendescribed, each of which transduces the redox signal tosecondary downstream effector proteins.

21. Aer — a redox sensor involved in aerotaxis • Aer regulatesthe motility behaviour of E. coli in gradients of oxygen(aerotaxis), redox potential and certain nutrients.Aerachieves this by interacting with the CheA–CheW complexto transmit sensory information to the flagellarmotors.

23. Pyridine nucleotides The pyridine nucleotides, NAD(H) and NADP(H), occupy central points in redox metabolism. The main role of NAD(H) is to shuttle electrons released during substrate oxidation to the electron transport chain. NADP(H) is involved in reductive biosynthetic and repair pathways.Considering the importance in maintaining redox balance of these cofactors it is perhaps surprising that sensors of their redox state have only recently been described.

24. Quinone redox sensors • ArcB — a regulator of respiratory gene expression The Arc two-component system of E. coli is a global regulator of gene expression under microaerobic and anaerobic growth conditions.When oxygen is limiting or absent, the membrane-bound sensor kinase ArcB autophosphorylates at His292 in the primary transmitter domain, then transphosphorylates the response regulator ArcA at Asp54.

26. Conclusions In vivo, neither redox sensors nor their signals operate in isolation. Integrated and complex regulatory networks provide an optimal response to changeable environments.With the advent of the post-genomic era, and the move towards more predictive biology, understanding how these complex regulatory circuits interact to integrate transcriptional responses to multiple environmental cues will be crucial if meaningful predictive model systems are to be developed.