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CO-Sensing Mechanisms

Department of Bacteriology, University of Wisconsin—Madison, Madison, Wisconsin

SUMMARY

INTRODUCTION

Sources and Biological Impacts of CO

Biological Utilization of CO

BIOLOGICAL CO SENSORS

General Features of CO Binding and Selectivity

CooA and CooA Homologs

Soluble Guanylate Cyclase

Neuronal PAS Domain 2

FixL

Other Potential CO Sensors

COOA AS A CO SENSOR AND RESPONDER

CooA Activity Assays

Insights into the Activation Mechanism from a Structural Comparison of CooA and CRP

Population Dynamics of the CooA Response to CO

Heme Vicinity: Structure and Implications

The Oxidation-Reduction Mechanism in CooA and Its Implications

Basis of the CO Specificity of R. rubrum CooA

Repositioning of C Helices as a Signal Transduction Mechanism

Achieving the Active Structure of CooA

Nature of the active form of CooA.

Interaction of CooA with specific DNA sequences.

Positioning CooA for proper interaction with RNA polymerase.

Intersubunit communication in CooA.

FUTURE DIRECTIONS AND OPEN QUESTIONS

ADDENDUM IN PROOF

ACKNOWLEDGMENTS

REFERENCES

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INTRODUCTION

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CO is a ubiquitous and globally increasing atmospheric pollutant (150 ppb) largely generated by the burning of fuels and biomass, with apparently minor contributions from numerous biological systems (23, 78, 132), which continue to be discovered (see, e.g., reference 25). Its removal occurs primarily through photochemical oxidation, along with significant degree of microbial utilization (23, 90). In humans, exposure to urban CO levels (ca. 100 ppm) and tobacco smoke may result in a variety of symptoms that are easily mistaken for viral illnesses (1, 134), while acute toxicity occurs at higher exposure, typically via the accumulation of combustion products in confined spaces with improper ventilation. As a result, some 2,000 non-fire-related CO-poisoning deaths occur annually in the United States (92). Human toxicity is attributed to the high affinity of CO for iron such that the oxygen capacity of hemoglobin is reduced, and the gas is broadly inhibitory for heme proteins and nonheme iron enzymes including hydrogenase (2, 45, 106) and nitrogenase (20, 83, 106). In light of its heme affinity, it is a biological irony that the primary endogenous source of CO in mammals is heme degradation catalyzed by multiple heme oxygenase activities in the cell. These heme oxygenases are differentially expressed and regulated, and they produce micromolar levels of CO in cell cultures (12). On average, humans produce 0.4 ml of CO per h. This endogenous CO production in mammals highlights the importance of the discrimination against CO by hemoglobin. Without such discrimination, endogenous CO production alone would result in 20% CO-bound hemeproteins (22).

Diverse aerobic carboxidotrophs express heterotrimeric Mo-containing enzymes that couple CO oxidation (CO + H₂O CO₂ + 2e^– + 2H⁺) to CO-insensitive respiration (87, 91). These enzymes characteristically demonstrate high affinity for CO, in part reflecting its reduced solubility at the thermophilic growth temperatures of several carboxidotrophs, and the oxidation is typically coupled to CO₂ fixation via the Calvin-Benson-Bassham reductive pentose phosphate cycle. CO-dependent expression has been shown, although the mechanism of this regulation remains undefined (113). Anaerobic or anaerobically cultivated members of the Archaea (methanogenic and sulfate reducing) and Bacteria (sulfate reducing, acetogenic, hydrogenogenic, and phototrophic) express (sometimes multiple) homodimeric, heterodimeric, and heteropentameric, and heteropentameric enzymes that catalyze reversible CO oxidation. These possess a Ni-containing C site, an activity readily assessed when coupled to dye reduction. Sequence analyses show remarkable protein conservation despite the diverse lineages (32, 74). In addition, the heteromeric enzymes catalyze an acetyl coenzyme A (acetyl-CoA) synthase (ACS) activity at the metallocluster A site, also containing Ni, that interconverts acetyl-CoA with CO, a cofactor-bound methyl group, and coA. The anabolic formation of acetyl-CoA represents the identifying feature of a widespread mechanism of carbon fixation termed the Wood-Ljungdahl pathway and has been suggested as a primordial anabolism (64). In addition, CODH-ACS catalyzes the reverse process during the anaerobic catabolism of acetate (34, 39, 40, 52, 104, 142).

It is important to note that (i) the CODH-ACS-catalyzed one- and two-carbon interconversion is a central function in anaerobic metabolism in which CO is a sequestered intermediate in the reaction and (ii) as a fundamental anabolic and/or catabolic anaerobic process, the bifunctional Ni-containing enzymes are expressed independently of exogenous CO (see. e.g., references 7 and 72). In contrast, expression of the monofunctional CODH enzymes can readily be associated with environmental CO: the anaerobic Carboxydothermus hydrogenoformans (Ni-containing CODH) was obtained from a volcanic vent (121), while the aerobic Streptomyces thermoautotrophicus (Mo-containing CODH) was isolated from soil covering mounds of burning charcoal (110).

Structures of both the Mo- and Ni-containing enzymes have been published recently. The carboxydotrophic enzymes isolated from Oligotropha carboxidovorans and Hydrogenophaga pseudoflava display hydrophilic and hydrophobic channels to the Mo- and Cu-containing reaction center and FeS centers appropriately spaced for electron transfer (30, 31, 49, 55). Published structures (26, 33) of the bifunctional enzyme from the anaerobe Moorella thermoacetica (formerly Clostridium thermoaceticum) also display a complement of FeS centers for electron transfer, as well as the A and C Ni-containing reaction centers connected by a 70-Å hydrophobic channel through which CO transits. The evident channeling, also indicated experimentally (86, 115), confirms the role of CO as a central metabolic intermediate despite its modest solubility and environmental paucity. Finally, structures of monofunctional Ni-containing enzymes from the thermophilic C. hydrogenoformans and the photosynthetic Rhodospirillum rubrum have been reported (32, 35). These are structurally similar to the ß-subunit of the M. thermoacetica enzyme and contain the CO-oxidizing C center. In R. rubrum, carbon fixation occurs via the Calvin-Benson-Bassham cycle or alternative mechanisms but not via acetyl-CoA synthesis (69).

Anaerobic, CO-dependent catabolism by phototrophs was first described in 1968 (63) and subsequently elaborated for a strain of Rhodopseudomonas (now Rubrivivax gelatinosis) (131) and R. rubrum (13, 37, 74, 132). The R. rubrum process, the subject of considerable studies in the laboratory of Paul Ludden, depends on the CO-induced, anaerobic expression of a monofunctional Ni-CODH, a CO-insensitive hydrogenase (41), associated electron transfer (42), and Ni-mobilization (68, 73, 138) components. Eleven identified genes, designated coo (for "CO oxidation"), are organized into two regulated transcripts. One encodes the hydrogenase subunits and associated components (cooMKLXUH), and the other encodes CODH (cooS gene product), an unusual FeS protein, and components for Ni storage and insertion (cooFSCTJ). Remarkably similar proteins (and genetic arrangement) are found in C. hydrogenoformans (118). In R. rubrum, energy derived from the thermodynamically marginal process (CO + H₂O – CO₂ + H₂, G^o' = –20 kJ/mol) depends on vectoral proton translocation, possibly by CooU, CooM, CooK (42), or CooX (4). Genes for similar CODH enzymes and overlapping sets of the auxiliary functions have been found in the sulfate-reducing Desulfovibrio vulgaris Hildenborough (137), Desulfovibrio desulfuricans (wherein Yagi first detected CODH activity [143]), and the aerobic nitrogen-fixing Azotobacter vinelandii. As described below, all of these organisms contain a gene homologous to cooA, whose product in R. rubrum senses environmental CO and initiates transcription of the CO-oxidizing/H⁺-reducing system. Descriptions of studies of CooA of R. rubrum form a major part of this review because of the following broad biological implications: (i) it is the clearest example of biological CO signal reception, a phenomenon recently proposed for mammalian systems as well (12), (ii) it is a prototypical heme-based sensor, and (iii) it serves as a model for the mechanism of action of other members of the CRP/FNR (cyclicAMP [cAMP] receptor protein/ fumarate nitrate reductase regulator) family of transcriptional regulators.

BIOLOGICAL CO SENSORS

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The existence of heme-containing CO sensors is itself unsurprising, since CO can bind to virtually all heme-containing proteins. This property means that all heme-containing proteins are possible candidates for being biological CO sensors even though they are known to have other obvious physiological gaseous ligands. CooA, the only CO sensor demonstrated to be physiologically relevant, and other candidate CO sensors have similar general properties to other heme-based sensors. As a group, heme-based sensors commonly exploit histidine as a proximal ligand. Typically, this Fe-His bond is weak, as evidenced by low Fe-His stretching frequency in the resonance Raman spectrum (126, 129), but the functional role of this weak bond is unclear. On the other hand, each heme-based sensor possesses a unique heme environment, which affects its specificity for different small-molecule ligands. There are three levels of ligand specificity in heme-based sensors. The first is at the level of binding, which is important for heme-based O₂ sensors because the natural affinity for O₂ is lower than that for CO by a factor of 20,000. In hemoglobin and myoglobin, the ratio is reduced to 25 to 200 (119) by exploiting H-bonding between the distal histidine side chain and O₂ ligand. Nitrophorins, NO transporters found in the saliva of the blood-feeding insect Rhodnius prolixus, use the unique NO binding to Fe(III) heme for ligand discrimination, to which state CO or CO₂ hardly binds (18). The second level of ligand specificity involves the coordination property of the ligand. This level is useful for NO sensors since NO uniquely exerts a strong trans effect, occasionally weakening the trans-ligand bond so much that a five-coordinate NO adduct results. This is important for the selectivity of soluble guanylate cyclase (sGC) for NO, as described below. The third level of selectivity is through allostery, which is unique in each heme-based sensor and represents the conformational change that the sensor undergoes in response to ligand binding. For a given sensor, the ligand specificity is likely to be provided by a combination of these three levels. For each of the following examples of sensors capable of binding CO, we briefly address what is known about the molecular basis for ligand discrimination.

View larger version (19K): [in this window] [in a new window]   FIG. 1. The general behaviors of CooA, sGC, NPAS, and FixL in response to their effectors. In all cases, the relevant heme pockets are depicted as boxes. (A) In the absence of CO, the CooA homodimer has its DNA-binding surfaces (shaded black) buried away from the solvent. On CO binding, there is a significant rearrangement of a portion of each monomer that allows the DNA-binding surfaces to bind specific DNA sequences. The DNA-bound CooA then interacts with RNA polymerase to allow gene transcription. (B) sGC exists as a heterodimer that is inactive without NO. NO binding to the heme displaces an endogenous histidine ligand (–H), which triggers a conformational change. The active conformation of sGC synthesizes cGMP, an important signal molecule. (C) NPAS2 is only recently described and poorly understood. In the presence of small molecules bound to the heme, the protein exists as an inactive monomer, but the absence of such small molecules leads to the formation of an active heterodimer with another protein, BMAL1, which is able to bind DNA and activate transcription. (D) FixL acts as a homodimer that is inactive when O2 is bound to the hemes. In the absence of O2, FixL undergoes a conformational change that causes autophosphorylation and subsequent transfer of that phosphate to FixJ. Phosphorylated FixJ binds DNA and activates transcription of anaerobically expressed genes.

CooA of R. rubrum is the prototype of a family of related proteins from a wide variety of bacteria that are apparently involved in CO sensing (147). This claim is based on the following arguments. (i) The CooA homologs all have a deletion of eight amino acids (with respect to the CRP sequence) that appears to provide space for the heme in CooA of R. rubrum. A number of other residues already shown to be critical for the functionality of CooA of R. rubrum are also conserved in the homologs. (ii) All of these homologs are found in genomes where there is also a gene for a CO dehydrogenase that is homologous to the NiFe CODH of R. rubrum, whose expression is regulated by CooA in that organism. (iii) The genes for six of these CooA homologs have been cloned in E. coli, and four allowed CO-dependent gene expression in the E. coli reporter system used for analysis of CooA of R. rubrum (147). (iv) The F helices of all the homologs, which serve to make specific DNA sequence contacts, are similar to each other, and there are appropriate palindromic sequence 5' of the genes for the CO dehydrogenases in each organism, although the actual biological function of these palindromes has not been experimentally demonstrated. Further discussion of the implications of these homologs for the general behavior of the CooA family is presented in the various appropriate sections that discuss CooA function in detail.

The structure of sGC is unknown, but its catalytic domain is thought to be composed of the C-terminal regions of the both subunits, which have sequence homology to regions in the particulate sGC and the adenylate cyclases. While both subunits are essential for catalytic activity, the N-terminal region of the ß subunit alone binds the heme prosthetic group (43). Not surprisingly, the presence of the heme prosthetic group is required for activation of sGC by NO (65) and NO has been shown to trigger a conformational change in sGC through the cleavage of the proximal histidine ligand, His105, resulting in five-coordinate high-spin NO adduct (150). The study of sGC by using a series of metalloporphyrins supports the hypothesis that it is the cleavage of the His-Fe bond that is critical (17, 28). For example, Mn(II)-containing protoporphyrin IX-reconstituted sGC binds NO but is not active, consistent with the role of the retention of the His-Fe bond. On the other hand, Ni(II)- or Cu(II)-containing protoporphyrin IX-containing sGC is active without NO. This latter result is easily rationalized by the fact that Ni(II) or Cu(II) protoporphyrin IX favors four-coordinate geometry, and therefore these reconstituted sGC variants lack the His-Fe bond. Probably by a similar mechanism, sGC with a free-base protoporphyrin IX, lacking an iron and therefore incapable of forming the His-Fe bond, is active. However, the observation that heme-free sGC is not active indicates that cleavage of the His-Fe bond is not sufficient and that the heme vicinity also plays a crucial role in the activation mechanism of sGC.

Recently, CO has been shown to inhibit platelet aggregation and promote the relaxation of vascular smooth muscle (15, 133), which are activities normally attributed to the action of NO on sGC. Consistent with the hypothesis that this CO activity reflects the activation of sGC, these CO activities have been shown to be cGMP dependent (15, 105). It has been known for some time that CO can enhance sGC activity in vitro, although its fourfold effect is much lower than that of NO and seems inconsistent with the degree of physiological response seen. Indeed, since CO binding to sGC leads to a six-coordinate low-spin heme adduct, with an intact His-Fe bond, the activation mechanism of CO must be different from that of NO. Importantly, however, the presence of YC-1 [3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole], a small synthetic molecule that has been found to enhance the CO effect in vitro, allows activation by CO to reach the same level as that of NO-stimulated sGC (44). The molecular basis for the stimulation mechanism of YC-1 to the CO-bound sGC is unclear, but YC-1 apparently does not force the cleavage of the Fe-His bond (75), as might be expected if it also led to a five-coordinate adduct. The question remains whether CO is a physiological ligand of sGC and if there is a natural product in vivo that mimics the stimulation caused by YC-1 in vitro. While there is no evidence available for the latter question, there are strongly suggestive data in support of the former. First, CO-induced vasorelaxation can be blocked by inhibiting sGC activity (46), consistent with a direct role in vivo for CO and sGC. Second, there is the striking evidence that heme oxygenase, the source of CO in nerve cells, colocalizes with sGC in cells with little or no nitric oxide synthase expression, supporting a link between CO and sGC (66, 135, 136). While the possible role of sGC in mammalian CO sensing is tantalizing but unproven, there is little doubt that some mammalian CO sensor must exist.

In the absence of O₂, FixL is autophosphorylated at an invariant histidine residue and the phosphoryl group is transferred to FixJ, leading to an enhancement in transcriptional activity. Under aerobic conditions, O₂ binding to the heme domain inhibits the histidine kinase activity in the C-terminal domain by 15-fold. The heme-binding domain of FixL proteins is a PAS domain (an acronym for the proteins with this domain, Per/Amt/Sim) (38, 122), a sequentially and structurally conserved motif that commonly serves as the sensor module of two-component signal transducers. In addition to FixL, two types of O₂-sensing phosphodiesterase, EcDos (27) and AxPDEA1 (19), contain PAS heme sensors.

Although the cognate physiological effector of FixL is certainly O₂, there is substantial disagreement in the literature about both the degree and the basis of ligand specificity. Many of the challenges in addressing this issue have recently been described (36, 128). The first issue is that many of the original hypothesis about ligand specificity were based on structures of only a portion of FixL and thus missed any effects of the rest of this protein or of FixJ, its regulatory partner. The second concern is that there are a number of biochemical properties of the protein that are altered in a variety of ways by the binding of different small-molecule effectors, so that the physiologically significant biochemical response remains unclear. Finally, the situation is complicated by the fact that FixL has been extensively studied from two different organisms, Bradyrhizobium japonicum and Sinorhizobium meliloti. However, the assays and the data sets for the two proteins are different, and it appears that their biochemical properties might be different as well. In the autophosphorylation assay, FixL of S. meliloti shows a broad ligand specificity in vitro (128). CO induces a conformational change, resulting in a 5-fold decrease of autophosphorylation activity of FixL, while NO causes a 2-fold decrease, CN^– causes a 15-fold decrease, and imidazole causes a >75-fold decrease. With FixL of B. japonicum, the more relevant "turnover" assay, which measures the phosphorylation of FixJ, shows rather more selectivity toward O₂.

Over the past few years, several hypotheses for activation, including the selectivity toward O₂, have been proposed. The first was the so-called spin-state hypothesis (47, 128), which was based on the observation that high-spin forms of FixL [Fe(III), Fe(II), and fluoro-FixL] have the same autophosphorylation activity and that low-spin forms inhibit the activity of FixL. Flattening of the heme induced by a switch of the iron atom from high spin to low spin by these ligands seemed to inactivate FixL reversibly. However, it now appears that not all low-spin forms are inhibited to the same degree, which is difficult to explain by this model, nor does it explain some recent mutational results (36). An alternative model is the "loop displacement hypothesis" which suggests that the critical element in inactivation is movement of a particular loop in the protein, termed the FG loop. While this model is attractive, the exact mechanism by which ligand binding effects this displacement remains obscure (36), although Arg220 (discussed below) probably plays a role.

The possible role of Arg220 as the trigger for the conformational change in B. japonicum FixL is based mainly on X-ray crystal structures. The heme-binding domains of most of the B. japonicum FixL structures (free or liganded form) have been solved, although these have not been in the context of the rest of the protein (50, 51, 56, 67) (Fig. 2). The binding of strong heme ligands changes the heme planarity in FixL and probably weakens the salt bridge between Arg220 and heme propionate 7 which is found in met-FixL of B. japonicum. The released Arg220 can move into the heme pocket and serve as a steric barrier that stabilizes the inactive FixL conformation. The greater sensitivity of FixL to O₂ and CN^– than that of other ligands can be explained by this model, since O₂ and CN^– are capable of holding the released Arg220 in the heme pocket. Nonetheless, another element must be responsible for the inhibition of the phosphorylation activity in CO-, NO-, and imidazole-bound forms of FixL, because in those structures, Arg220 movement into the pocket has not been observed (67).

View larger version (40K): [in this window] [in a new window]   FIG. 2. Current model for the behavior of the heme vicinity of FixL in response to O2 binding. (A) Active ferrous FixL (Protein Data Bank no. 1LSW), where the heme is depicted at the center, with the Fe atom as the dark sphere. H200 is the endogenous ligand in this five-coordinate form. (B) On O2 binding (Protein Data Bank no. 1DP6), there is a slight movement of the Fe atom with respect to the heme but a substantial movement of heme pocket residues. The movement of R220 (darkened in the figure) is thought to be particularly significant and results in a repositioning of the heme proprionates with respect to other residues in the heme pocket.

One might wonder why FixL shows broad ligand specificity, although it might reflect an inherently stronger affinity of free heme for NO or CO than for O₂. This challenge of competition by physiologically inappropriate small molecules is partly overcome in myoglobin through the use of polar distal heme pocket residues (most importantly a histidine residue) which preferentially stabilize O₂ over CO. However, a similar mechanism does not appear to be employed by FixL, since its distal heme pocket is composed of highly hydrophobic amino acids (Ile209, Leu230, and Val232 in S. meliloti FixL; Ile215, Leu236, and Ile238 in B. japonicum FixL). The uncertainties about the role of CO in FixL function will probably be resolved in the near future.

sGC has been described as a possible CO sensor, but other eukaryotic CO sensors might exist. There is emerging evidence suggesting that at least some CO effects are mediated through a cGMP-independent, mitogen-activated protein kinase pathway. Anti-inflammatory effects and the antiapoptotic action of CO are good examples (14, 97). The precise mechanism by which CO might activate the kinases remains to be elucidated, and the target protein of CO is not yet identified, but further studies might lead to the identification of eukaryotic CO sensors.

COOA AS A CO SENSOR AND RESPONDER

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CooA of R. rubrum is not only the best understood heme-containing sensor but also highly specific in its response to CO as an effector. As discussed below, CO binding to the reduced heme of CooA causes a substantial conformational change in the protein that enables it to bind specific DNA sequences and thereby activate transcription. We first give some background about the activity and structural features of CooA and then address what is known about CO binding and its response.

The sequences bound by CooA are reminiscent of those bound by CRP and FNR, which is consistent with the relatively high similarity among the F helices of these proteins (see below), which make the specific base contacts. For proper biological regulation of expression, the affinity of the binding site for the activator should be such that there is very little occupancy of the site in the absence of the effector but very high occupancy in the presence of the effector.

CooA has been routinely examined for its activity in vivo, using reporter systems that measure the ability of CooA to bind a specific DNA sequence and then properly interact with RNA polymerase to activate transcription and produce a product that can be assayed. When analyzing CooA activity in R. rubrum, the assay has typically been the activity of the CODH itself, but this is somewhat indirect, since that activity is a reflection of not only gene expression but also CODH maturation (73, 138). There are several technical advantages in using an E. coli reporter strain with lacZ fused to one of the two normal CooA-responsive promoters from R. rubrum. When cooA is expressed from a plasmid, this strain expresses very low ß-galactosidase activity unless the cells are anaerobic and exposed to CO. Such an assay shows a linear response over a certain range of CooA activity, because maximal ß-galactosidase activity requires only that the CooA-binding site upstream of lacZ be saturated (71). Significant differences in the fraction of the CooA population in the active form can therefore be missed unless total CooA levels are tuned through regulation of the cooA promoter.

While more time-consuming, an in vitro assay of DNA binding by CooA has significant advantages. The most readily interpretable assay involves fluorescence anisotropy, in which a fluorescently tagged DNA fragment containing the CooA binding site is incubated with purified CooA (125). This assay can provide a K_d for any tested variant in the presence and absence of effector. It measures only DNA affinity, rather than the complex combination of DNA affinity and affinity for RNA polymerase that is measured in vivo. Other assays such as gel shifts (D. Shelver and G. P. Roberts, unpublished data), footprinting (117), and in vitro transcription assays (59) have also been successfully applied to CooA.

View larger version (46K): [in this window] [in a new window]   FIG. 3. Comparison of the structures of active CRP and inactive CooA. (A) Active CRP (Protein Data Bank no. 1G6N) is a symmetrical homodimer that is rotated slightly here to display critical features. The two monomers are colored differently, and the upper portion of each constitutes the DNA-binding domain, although this is difficult to see in this protein species. The F helices, which make specific DNA contact, are depicted in yellow, and other important helices are indicated. The approximate positions of the AR1 and AR2 regions are indicated on onemonomer, and the AR3 region is circled on the other. cAMP is indicated by the pair of the ball-and-stick molecules near the center of each monomer. (B) The X-ray crystal structure of inactive CooA (Protein Data Bank no. 1FT9) shows that this protein exists in two forms, with the more extended form termed form B, although in each form the F helices are buried away from the solvent. DNA- and effector-binding domains are roughly indicated on the right side of the panel. Because of the extended structure, the AR3 region, at the tip of the ß-4/5 loop, is easily seen. The heme region is depicted as the ball-and-stick structure and enlarged in panel C. (C) The heme vicinity of one CooA monomer is shown, with nearby residues noted and identified as to the protein monomer in which they are found: (a) refers to the monomer on the left, while (b) refers to the monomer on the right. His77 is shown as the ligand in the ferrous form, and Cys75; the ligand in the oxidized form, lies immediately behind it in this view. The other ligand, Pro2, is the N terminus of the right protein monomer and is connected to the red chain at the bottom right of this panel.

Comparison of the DNA-binding domains of CRP and CooA shows that the individual domain structures are remarkably similar to each other (81), at least for regions that are resolved in each structure. However, it is the positioning of these domains with respect to each other and to the effector-binding domains that is very different in CooA and CRP. Irrespective of which of the two forms of inactive CooA is more similar to the solution form of the protein, they have important similarities: compared to the structure of active CRP, both forms are relatively elongated and have a dramatic repositioning of the DNA-binding domains, such that the F helices are actually turned away from the solvent. This latter point is consistent with the NMR analysis of inactive CRP noted above (141). These results lend credence to the hypothesis that CRP and CooA might undergo a roughly similar conformational change during activation. Effector binding must in some way signal a significant change in orientation of the DNA-binding domains in each protein. The signal transduction pathway that effects this change is of central importance to understanding these proteins.

Within the effector-binding domains, the obvious difference is the heme of CooA. The next most obvious difference between CRP and CooA is in the position of the 4/5 loop within each structure; the 4/5 loop refers to a pair of ß strands that extend from the effector-binding domain in each protein toward the DNA-binding domain. While this change in position cannot be definitively attributed to the activation process, it is a very reasonable hypothesis, since the position of that loop in each protein predicts different contacts with the DNA-binding domains, presumably stabilizing each structure. Because the repositioning of the DNA-binding domains is certainly relevant to the activation process, it follows that surfaces within the effector-binding domain that contact the DNA-binding domain in either the active or inactive form of the proteins might also be repositioned after effector binding, a notion that is also addressed below where the protein surfaces that interact with RNA polymerase are discussed. It therefore seems highly likely that there are specific conformational changes within the effector-binding domains on effector binding. However, the actual nature of these structural changes remains poorly understood, except for those in the immediate vicinity of the cAMP in CRP and the heme vicinity in CooA.

The repositioning of the two effector-binding domains with respect to each other on effector binding is clearly central to the process of activation. This notion was first demonstrated by the Poulos group, who solved the effector-free CooA structure and compared it to that of effector-bound CRP (81). They noted a modest change in the relative position of the two long helices, termed the C helices, that lie at the dimer interface of the two proteins. They suggested that this C-helix repositioning might serve as a signal transduction pathway between the heme region of CooA and the DNA-binding domains. As detailed below, this hypothesis has been strongly supported by direct mutational analysis of CooA. A similar notion was also proposed for CRP, where cAMP binding immediately adjacent to these helices might cause their repositioning (100).

In conclusion, a structural comparison of inactive CooA with active CRP shows that there is a substantial change in the position of the DNA-binding domains of CooA after CO binding and that repositioning about the C helices is a likely factor in that response. The data are also consistent with the notion that CRP and CooA undergo similar conformational changes on effector binding, but this speculation requires substantially more experimental testing. Understandably, the basis of CO recognition by CooA therefore requires an analysis of why CO leads to such a repositioning whereas other small molecules do not. This is addressed below.

Another complication involving a equilibrium between different protein forms probably exists with inactive CooA. In both forms of inactive CooA in the known X-ray structure, the F helices are buried from the solvent. However, one would expect that each of these regions would have some low affinity for a variety of DNA sequences. One might then predict that the very high concentration of DNA found in the cell might perturb the CooA structure by interaction with one or both of the F helices in a nonspecific way. If it is true that the actual structure of inactive CooA in the cell has a different arrangement of the DNA-binding domains from that depicted in the known structure, we will have difficulty in understanding which interactions stabilize and destabilize that structure. This would also be relevant to the analysis of the response of CooA to CO, since it would affect the actual nature of the CooA population that senses CO and therefore the pathway of CO activation.

His77 serves as one heme ligand in reduced CooA and is critical for the response of CooA to CO, because substitution with any other residue at that position destroys the CO-dependent response of the protein (116; M. Conrad, H. Youn, and G. P. Roberts, unpublished data). His77 is important for two reasons. First, the His ligation is at a critical poise of ligand strength. It must be sufficiently strong to avoid displacement by CO or other small molecule ligands, since such binding to the "wrong side" of the heme does not allow proper C-helix repositioning. However, it must also not be so strong that a six-coordinate NO adduct is formed, since such a species might well be active. The second important property of His77 is that its serves as the tether to the CO-bound heme. Its precise size and positioning are therefore important for the precise positioning of the CO-bound heme with respect to the C helices, and this last interaction is important for activation of CooA. The ligand strength and positioning of His77 must also be relevant to the proper redox-mediated ligand switch between Cys75 and His77 that is described immediately below. It is therefore not surprising that all CooA homologs have a His residue at the homologous position of His77 (Fig. 4).

View larger version (88K): [in this window] [in a new window]   FIG. 4. Sequence alignment of the CooA homologs (reprinted from reference 147). This shows the sequence comparison of the CooA homologs described in the text, with CooA of R. rubrum shown at the top and, for comparison, the CRP of E. coli at the bottom. Residues that are extremely or modestly conserved among the CooA homologs are shaded in black and gray, respectively. Above the top line, specific -helix or ß-sheet regions are noted, as are the following important regions: the distal heme pocket, which is formed both by the N-terminal residues and those around positions 112 to 117 in R. rubrum CooA (dotted lines); the proximal heme pocket, including Cys75 and His77 (dotted lines); the ß-4 and ß-5 regions forming the 4/5 loop and flanking the AR3 region; the portion of CooA deleted relative to CRP (termed "Gap"), which provides space for the heme; the hinge region with Phe132, which separates the effector- and DNA-binding domains; AR1, by analogy to a critical residue in CRP; and the E and F helices, which define the DNA-binding region, with Gln178 shown near the beginning of the F helix. Rr, R. rubrum; Dh, D. hafniense; Ch, C. hydrogenoformans; Av, A. vinelandii; Dd, D. desulfuricans; Dv, D. vulgaris; Ec, E. coli.

Asn42 is the other proximal-side residue (in addition to His77) that is directly perturbed when CO binds to the heme. This residue makes H-bonding contacts with His77 in the reduced form but not in the CO-bound form (24, 81). Since His77 is tethered to the heme in both forms, this structural change of His77 suggests a repositioning of His77 with respect to Asn42 in response to CO. CooA variants with substitutions at this position are somewhat perturbed in their ability to be activated by CO, but the precise basis for this is not clear (24). It is also interesting that the adjacent residue is Glu41, which has an effect on CooA-RNA polymerase interactions (82). This suggests that CO binding might have a direct effect on this interaction as well, as discussed below.

The other ligand in the reduced form of CooA, Pro2, is the N terminus of the other protein monomer (81). Proline had not previously been detected as a heme ligand in any protein because it is sterically incapable of playing that role except when it happens to be at the N terminus. The presence of such a novel ligand immediately suggested that it might be critical for the proper activation of CooA, but mutational analysis has disproved that (125) and has shown that a variety of substitutions at this position provide substantial CooA activity. This view is supported by the observation that none of the other CooA homologs appears to have a proline positioned to serve as a ligand (Fig. 4). However, while Pro2 is not critical for the function of CooA in R. rubrum, it does appear to be optimal.

Another residue is important in stabilizing Pro2 as a ligand, and that is Arg4, which appears to interact with a propionate of the heme (81). Removal of this residue by deletion results in a detectable population of five-coordinate heme in both the oxidized and reduced forms of CooA (125).

When CO binds to the heme of CooA, it replaces one of the two protein ligands, but the identity of the displaced ligand was unknown for some time. In part this reflected the fact that there was no spectroscopic data set for the novel Pro ligand to serve as a control for the CO-bound form of CooA. The issue was resolved by the application of NMR by the Aono group, which showed that CO replaces Pro2 (144). A resonance Raman analysis has indicated that the displaced Pro2 is not in the immediate vicinity of the bound CO (24). This result is consistent with the observation that alteration of Pro2 in CooA does not dramatically impair the ability of the protein to achieve the active conformation (125). However, there appear to be three auxiliary roles for Pro2 in R. rubrum CooA function. The first is that its ligation to the heme helps keep the protein in the inactive form until CO binds. In an otherwise wild-type background, alteration of Pro2 does not yield a substantial increase in CO-independent activity, which would be the expected result if Pro2 ligation were critical for this role (125). However, an involvement of Pro2 in this process is revealed in backgrounds with other substitutions that enhance CO-independent activity and in which the replacement of Pro2 is synergistic for this response (71). We assume that the modest effect seen in an otherwise wild-type background is because of the presence of other unidentified protein ligands that can adequately maintain the inactive form. The second role of Pro2 is that it provides a heme ligation that is weak enough to be displaced by CO yet strong enough to resist displacement by weaker small-molecule ligands. It is not clear if the residues that replace Pro2 in variants of R. rubrum CooA or in the CooA homologs have similar properties, because binding of other small molecule has not been examined with these proteins. Finally, Pro2 and its adjacent N-terminal residues must be flexible enough to remain ligated to the heme through the oxidation-reduction process. As explained in the following section, this process involves a significant movement of the heme relative to the protein, requiring ligand flexibility.

In the CO-bound form of CooA, the residues presumed to be near the heme-bound CO are all from the two C helices at the dimer interface. These include Leu112, Ile113, Leu116, Gly117, and Leu120 (146). The evidence for the rolles of these residues is presented below, but some general comments are appropriate here. It is important to recognize that the structure of the CO-bound form of CooA is unknown, and so the exact position of the CO-bound heme with respect to amino acid residues is not clear. Despite these uncertainties, a number of CooA variants altered at positions 113, 116, and 117 show perturbations in the C—O and Fe—C stretching frequencies as determined by resonance Raman spectroscopy (24). This suggests that the bound CO is located near these residues. As detailed below, some of these residues are critical for the activation of CooA in response to CO. Other substitutions in this region create CooA variants that respond effectively to imidazole as an effector (146; H. Youn, R. L. Kerby, and G. P. Roberts, unpublished data), consistent with a role of this region in interacting with the small molecule bound to the heme. In the CooA homologs, Leu116, Gly117, and Leu120 are all strictly conserved while positions 112 and 113 have conservative substitutions (Fig. 4). These results are consistent with the hypothesis that it is the interaction of this portion of the protein with the CO-bound heme that leads to the repositioning of the C helices in the normal activation process.

In summary, there are two obvious local changes in the vicinity of the CooA heme in response to CO: displacement of proximal Pro2 ligand, which allows repositioning of the CO-bound heme with respect to the C-helix residues, and breakage of the His77-Asn42 H-bond. This combination of features in the unique CooA heme-binding motif ensures that only CO can trigger the structural rearrangement necessary for activation.

View larger version (41K): [in this window] [in a new window]   FIG. 5. Structure of the heme in the reduced form of CooA, oriented to show the relative positions of Cys75, which serves as the ligand in the oxidized form, and His77, which serves as the ligand in the reduced form. The Cys75 and heme Fe must move 2 to 3 Å with respect to each other for this ligation to occur.

A number of interesting and biologically significant questions concerning the redox switch in CooA remain unanswered. One is obviously the exact nature of the conformational change that occurs within the effector-binding domain to not only allow this switch but also stabilize both forms of the protein. At present, we have relatively little insight into the homogeneity of either of these species. Indeed, the original analysis of CooA redox properties showed a curious hysteresis such that the curves obtained for oxidation were distinct from those obtained for reduction. This behavior was rationalized by a very slow interconversion between the two forms (94), yet a different analysis by the same group revealed that the conversion occurred in the millisecond range (93). The basis for this discrepancy is unknown. A second question concerns the identity of the ligand trans to Cys75 in oxidized R. rubrum CooA. Indirect evidence also suggests that Pro2 serves as the ligand trans to Cys75 in the oxidized form of CooA (125, 145). Finally, a more biologically interesting question concerns the actual chemical entities sensed by CooA for this redox transitional, though O₂ can certainly suffice. Presumably it is some pool of small molecules such as NAD and NADH, but the identity of that small molecule remains unknown.

We have already explained that the strength of the endogenous protein heme ligands can explain the remarkable CO specificity of R. rubrum CooA for its activation. The simple hypothesis was that only CO could form a six-coordinate species by displacing the Pro2 ligand and that this form might therefore be both necessary and sufficient for activation. The obvious prediction was that perturbation of the Pro2 ligation could weaken that bond and allow other small molecules to bind the heme on the proper side. This happens to be true, based on the following analysis of the P3R4 variant of CooA, in which the codons for the third and fourth residues have been deleted. This alteration eliminates the Arg4 residue that stabilizes Pro2 ligation to the heme, producing a small but significant population of five-coordinate heme in the reduced form. Not surprisingly, this variant is able to bind CN^– and imidazole very efficiently, but binding of these molecules does not activate the protein to a detectable extent (146). This result disproves the simple hypothesis above and indicates that there is another level of discrimination for CO. What might be the basis for this discrimination, especially against CN^–, which is so similar to CO in size?

It is clear that the bound CO exists in a very confined pocket in R. rubrum CooA, because rebinding of CO after its removal by photolysis is unusually rapid and efficient (5, 112, 130). Because the structure of the active form has not been solved, the identity of this pocket is unknown, but it is apparently not formed by the N terminus, as evidenced by the resonance Raman results cited above. It is therefore presumed that the pocket must be formed by the only other residues in the heme vicinity, which are those on the C helices of both protein monomers. The nature of this pocket is of interest for two biological reasons that are explained further below. First, the interaction of the CO-bound heme with the C helices is almost certainly a critical step in signal transduction within CooA since it causes the C-helix repositioning necessary for activation. Second, as described immediately below, the nature of the interactions in this heme pocket must certainly play an important role in the specificity of the CO response.

Under the hypothesis that this CO specificity results from a precise interaction between the CO-bound heme and the C-helix residues, a number of these have been analyzed by randomizing the codons singly or in small groups and then screening for variants that responded to CO. The expectation was that certain positions should be critical for a response to CO. The presumption that these residues were in the general vicinity of the bound CO was supported by the observation that certain substitutions at positions 113, 116, and 117 (Fig. 3C) perturbed the CO stretching frequency in resonance Raman analysis (24). In fact, only Gly117 was absolutely required for a CO response, although position 120 was also fairly stringent, tolerating only Ile in place of Leu120 (146, 147; R. L. Kerby and G. P. Roberts, unpublished data). While it is tempting to suppose that these might be the residues that determine CO specificity, this hypothesis has been weakened by the results presented below for CooA variants that respond to imidazole.

In a similar analysis, a variety of hydrophobic residues were found to be acceptable at positions 112, 113, and 116. However, the analysis provided the important observation that hydrophilic residues at these positions cause a decrease in the accumulation of heme-containing CooA and were also unable to respond to CO (24, 146). The first effect is consistent with the idea that a hydrophobic pocket is typically found around hemes and presumably serves to maintain the heme in the protein. The second result suggested the following hypothesis to explain why CO binding to the heme might lead to C-helix repositioning. CO binding to the heme displaces the Pro2 and its attached N terminus, which apparently moves away from the heme. This then exposes the largely hydrophobic surfaces of the C helices to an aqueous environment. The repositioning of the C helices that results in activation might then be the result of an effort to reduce the solvent exposure. Alternatively, hydrophilic residues at these positions might interfere directly with the proper C-helix positioning or indirectly by affecting heme positioning. The result of the above analysis was to suggest that Gly117 and Leu120 might make critical contacts with the bound CO, where the other residues were less likely to do that because a variety of hydrophobic residues at those positions allowed a fairly normal response to CO (146).

Concurrent with the analysis of the C-helix requirements for a proper response to CO, we analyzed the same region of CooA for its ability to allow activation by imidazole. Recall that the P3R4 CooA variant is able to bind CN^– and imidazole but is not activated by them. Under the assumption that the additional level of ligand specificity probably was due to residues in the vicinity of the bound ligand, we therefore started with P3R4 CooA, randomized various C-helix residues, and screened for activation in response to imidazole in vivo. Randomization of positions 117 and 120 yielded no imidazole-responsive variants, but the simultaneous randomization of position 113 and 116 did (Youn et al., unpublished). A variety of combinations of residues at these positions supported this phenotype, but the striking commonality was the presence of a Trp residue at one of the two positions. The basis for this is unknown, and it is clear that other aromatic residues are much less effective. The majority of the imidazole-responsive variants continued to be activated by CO as well, but some, such as P3R4 Trp113 Trp116 and P3R4 Arg113 Trp116, were substantially more active in response to imidazole than in response to CO. This result shows that these positions are critical for the imidazole response, presumably by some interaction with the bound imidazole itself, although more complicated mechanisms cannot be ruled out.

One imidazole-responsive CooA variant (P3R4 Trp113 Trp116 CooA) was then further analyzed for the importance of Gly117 and Leu120. The rationale was that if either of these residues provided a precise contact with the bound CO or, in a related way, served as the basis for CO specificity, then the requirements at these positions would be very different for a response to imidazole. In each case, only the wild-type amino acid residues were acceptable at these positions. While this does not disprove the notion that these residues make specific contacts with the heme-bound CO in wild-type CooA, it is much simpler to imagine that there are similarities in imidazole and CO responsiveness and that these residues are both critical in that shared pathway. The nature of the shared pathway would probably be the C-helix repositioning described below.

While it is therefore obvious that there is another level of CO specificity in CooA, the molecular basis for it remains unclear. Our current hypothesis is that the CO-bound heme must move to a hydrophobic region along the C helices and that this movement is precluded by ligands other than CO. Imidazole is both bulky and hydrophilic, so that its movement into such a pocket is prevented, while the charge on CN^– would also prevent its presence in a hydrophobic pocket. However, if imidazole is too bulky for normal activation, what is the basis of the imidazole-responsive variants that have been detected? Obviously the exact nature of the active forms of these variants is unclear, but our working hypothesis is that their precise mechanism of activation is different from that of wild-type CooA in response to CO. In other words, we imagine that the imidazole-bound heme interacts in a different way with the modified C-helix residues from the way in which CO interacts with the normal residues but that these different interactions both have the common result of C-helix repositioning. We then imagine that residues 117 and 120 are involved in that shared pathway. This result with the imidazole responders is particularly interesting since it indicates that CooA and its variants sense CO and imidazole by mechanistically different processes. In contrast, the models explaining the response of FixL to different small molecules assume that the sensing system of the protein is essentially identical for each effector (56).

A recent observation is also consistent with heme movement. Kinetic analysis has revealed that CO-bound wild-type CooA is heterogeneous in terms of the CO off-rate (103). One population shows a very low off-rate, consistent with the tight CO pocket already reported (112, 130). However, a roughly comparable population displays a significantly higher off-rate, implying a different position of the CO-bound heme. These two populations are in slow equilibrium, suggesting that a substantial conformational change might be occurring in the transition. This result is consistent with the notion that the two populations detected by this method might reflect the populations of active and inactive CO-bound CooA described above.

While we do not know the precise position of the CO-bound heme in CooA, it remains a tantalizing possibility that on CO binding, the heme approaches the position occupied by cAMP in active CRP. If this is correct, then the two proteins might be responding to their respective effectors in fundamentally similar ways. Determination of the mechanistic similarities and differences between the two proteins continues to be a focus of research because it should reveal commonalities for other members of the family of related proteins as well.

CooA, together with its homologs and also CRP and FNR, has a leucine zipper motif in the paired C helices. However, an analogous heptad repeat in the leucine zipper of all of these proteins, which lies about one-third of the way down the helices from the hinge region (positions 121 to 126 of CooA), is poor in comparison to a leucine zipper consensus. This led to the hypothesis that this nonconsensus heptad permitted flexibility in the structure, allowing a transition between an active and inactive form. Support for this notion for CRP has been made on structural grounds (100), and it is interesting that the D154A substitution that allows FNR to be active under aerobic conditions also affects this region (76). We reasoned that if helix repositioning was the signal pathway for CooA, then creating such a repositioning by mutation should short-circuit the signal and provide effector-independent activity. We therefore randomized the codons for positions 121 to 126 in an otherwise wild-type CooA background and screened for CO-independent variants (71). Sixty variants were sequenced, displaying a variety of different phenotypes, but all variants with substantial CO-independent activity had Leu residues (or other appropriate residues for a leucine zipper) at positions 123, 124, and 126. This is a fairly clear result and identifies helix repositioning as a major signal pathway within CooA. As noted above, the notion has also been proposed for CRP (100).

CooA variants with improved leucine zippers have substantial activity without CO but also show a further increase in activity, to approximately the wild-type level, in the presence of CO (71). Apparently that repositioning of the C helices is only partially effective at shifting the equilibrium to the active form if CO was not bound to the heme. There are two general possibilities to explain this. First, in the absence of CO, the improved leucine zipper variants might be under competing forces, with the continued Pro2 ligation to the heme preventing a full and proper repositioning. Second, CO binding to the heme might cause other conformational changes within the effector-binding domain that also assist in activation of the protein. In other words, while the C-helix repositioning is very important, it might not be the only signal pathway. In fact, both of these possibilities appear to be true.

The apparent tension caused by the retained Pro2 ligation was shown as follows. Among the CooA variants randomized at positions 121 to 126, one of the most active without CO had Ala121 and Gly122 substitutions. We noted that these substitutions lie between the improved leucine zipper region and the region of the C helices that are near the heme. These substitutions might therefore create a bend or a flexible region in the C helix, reducing the adverse tension in the absence of CO. Consistent with this, when the same pair of residues were introduced into an otherwise wild-type CooA background, the response to CO was diminished. This is reasonable because it is the rigidity of the helices in wild-type CooA that should be necessary for signal transduction through the protein. Subsequent analysis of that vicinity of the C helices is consistent with this idea, although there are contacts with other parts of the protein that complicate the analysis (71). A different confirmation of this model involved the addition of the P3R4 substitution to the improved leucine zipper background. By itself, the P3R4 causes negligible CO-independent activity in vivo, presumably in part because an adventitious ligand is able to satisfactorily replace Pro2 in keeping CooA inactive without CO. However, in the improved leucine zipper background, P3R4 allows very high CO-independent in vivo activity. This is easily rationalized by the fact that this variant can no longer efficiently tether Pro2 to the heme (nor can the adventitious ligand do this with the same effectiveness as Pro2) and therefore cannot effectively interfere with the C-helix repositioning caused by the improved leucine zipper.

The second possibility, that CO binding sends activation signals by other mechanisms, also has some support. As described below, there is good evidence that CO binding directly alters the positioning of some of the reg