The heme pocket afforded by Gly117 is crucial for proper heme ligation and activity of CooA.

CooA, a CO-sensing homodimeric transcription activator from Rhodospirillum rubrum, undergoes a conformational change in response to CO binding to its heme prosthetic group that allows it to bind specific DNA sequences. In a recent structural study (Lanzilotta, W. N., Schuller, D. J., Thorsteinsson, M. V., Kerby, R. L., Roberts, G. P., and Poulos, T. L. (2000) Nat. Struct. Biol. 7, 876-880), it was suggested that CO binding to CooA results in a modest repositioning of the C-helices that serve as the dimer interface. Gly(117) is one of the residues on the C-helix within 7 A of the heme iron on the Pro(2) side of the heme in CooA. Analysis of a series of Gly(117) variants revealed altered CO-sensing function and heme ligation states dependent on the size of the substituted amino acid at this position; bulky substitutions perturbed CooA both spectrally and functionally. A combination of spectroscopic and mutagenic studies showed that a representative Gly(117) variant, G117I CooA, was specifically perturbed in its Pro(2) ligation in both Fe(III) and Fe(II) forms, but comparison with other CooA variants indicated that perturbation of Pro(2) ligation is not the basis for the lack of CO response in G117I CooA. These results have led to the hypothesis that (i) the heme and the C-helix region move toward each other following CO binding and the interaction of the heme with the C-helix is crucial for CooA activation, and (ii) this event occurs only when a properly sized heme pocket is afforded.

CooA: a heme-containing regulatory protein that serves as a specific sensor of both carbon monoxide and redox state.

CooA, the heme-containing carbon monoxide (CO) sensor from the bacterium Rhodospirillum rubrum, is a transcriptional factor that activates expression of certain genes in response to CO. As with other heme proteins, CooA is unable to bind CO when the Fe heme is oxidized, consistent with the fact that some of the regulated gene products are oxygen-labile. Upon reduction, there is an unusual switch of protein ligands to the six-coordinate heme and the reduced heme is able to bind CO. CO binding stabilizes a conformation of the dimeric protein that allows sequence-specific DNA binding, and transcription is activated through contacts between CooA and RNA polymerase. CooA is therefore a novel redox sensor as well as a specific CO sensor. CooA is a homolog of catabolite responsive protein (CRP), whose transcriptionally active conformation has been known for some time. The recent solution of the crystal structure of the CO-free (transcriptionally inactive) form of CooA has allowed insights into the mechanism by which both proteins respond to their specific small-molecule effectors.

Mapping CooA.RNA polymerase interactions. Identification of activating regions 2 and 3 in CooA, the co-sensing transcriptional activator.

CooA is a CO-sensing protein that activates the transcription of genes encoding the CO-oxidation (coo) regulon, whose polypeptide products are required for utilizing CO as an energy source in Rhodospirillum rubrum. CooA binds to a position overlapping the -35 element of the P(cooF) promoter, similar to the arrangement of class II CRP (cAMP receptor protein)- and FNR (fumarate and nitrate reductase activator protein)-dependent promoters when expressed in Escherichia coli. Gain-of-function CooA variants were isolated in E. coli following mutagenesis of the portion of cooA encoding the effector-binding domain. Some of the mutations affect regions of CooA that are homologous to the activating regions (AR2 and AR3) previously identified in CRP and FNR, whereas others affect residues that lie in a region of CooA between AR2 and AR3. These CooA variants are comparable to wild-type (WT) CooA in DNA binding affinity in response to CO but differ in transcription activation, presumably because of altered interactions with E. coli RNA polymerase. Based on predictions of similarity to CRP and FNR, loss-of-function CooA variants were obtained in the AR2 and AR3 regions that have minimal transcriptional activity, yet have WT-like DNA binding affinities in response to CO. This study demonstrates that WT CooA contains AR2- and AR3-like surfaces that are required for optimal transcription activation.

Redox-mediated transcriptional activation in a CooA variant.

CooA, the carbon monoxide-sensing transcription factor from Rhodospirillum rubrum, binds CO at a reduced (Fe(II)) heme moiety with resulting conformational changes that promote DNA binding. In this study, we report a variant of CooA, M124R, that is active in transcriptional activation in a redox-dependent manner. Where wild-type CooA is active only in the Fe(II) + CO form, M124R CooA is active in both Fe(II) + CO and Fe(III) forms. Analysis of the pH dependence of the activity of Fe(III) M124R CooA demonstrated that the activity was also coordination state-dependent with a five-coordinate, high-spin species identified as the active form and Cys(75) as the retained ligand. In contrast, the active Fe(II) + CO forms of both wild-type and M124R CooA are six-coordinate and low-spin with a protein ligand other than Cys(75), so that WT and Fe(III) M124R CooA are apparently achieving an active conformation despite two different heme coordination and ligation states. A hypothesis to explain these results is proposed. This study demonstrates the utility of CooA as a model system for the isolation of functionally interesting heme proteins.

Structure of the CO sensing transcription activator CooA.

CooA is a homodimeric transcription factor that belongs to the catabolite activator protein (CAP) family. Binding of CO to the heme groups of CooA leads to the transcription of genes involved in CO oxidation in Rhodospirillum rubrum. The 2.6 A structure of reduced (Fe2+) CooA reveals that His 77 in both subunits provides one heme ligand while the N-terminal nitrogen of Pro 2 from the opposite subunit provides the other ligand. A structural comparison of CooA in the absence of effector and DNA (off state) with that of CAP in the effector and DNA bound state (on state) leads to a plausible model for the mechanism of allosteric control in this class of proteins as well as the CO dependent activation of CooA.

Characterization of variants altered at the N-terminal proline, a novel heme-axial ligand in CooA, the CO-sensing transcriptional activator.

CooA, the carbon monoxide-sensing transcription factor from Rhodospirillum rubrum, binds CO through a heme moiety resulting in conformational changes that promote DNA binding. The crystal structure shows that the N-terminal Pro(2) of one subunit (Met(1) is removed post-translationally) provides one ligand to the heme of the other subunit in the CooA homodimer. To determine the importance of this novel ligand and the contiguous residues to CooA function, we have altered the N terminus through two approaches: site-directed mutagenesis and regional randomization, and characterized the resulting CooA variants. While Pro(2) appears to be optimal for CooA function, it is not essential and a variety of studied variants at this position have substantial CO-sensing function. Surprisingly, even alterations that add a residue (where Pro(2) is replaced by Met(1)-Tyr(2), for example) accumulate heme-containing CooA with functional properties that are similar to those of wild-type CooA. Other nearby residues, such as Phe(5) and Asn(6) appear to be important for either the structural integrity or the function of CooA. These results are contrasted with those previously reported for alteration of the His(77) ligand on the opposite side of the heme.

Altering the specificity of CooA, the carbon monoxide-sensing transcriptional activator: characterization of CooA variants that bind cyanide in the Fe(II) form with high affinity.

CooA is a carbon monoxide- (CO-) sensing homodimeric heme protein that activates the transcription of genes required for the anaerobic oxidation of CO to CO(2) in the phototrophic bacterium Rhodospirillum rubrum. In this study, we demonstrate that mutational alteration of the histidine residue (His(77)) that serves as a heme ligand in the Fe(II) form of CooA allows high-affinity binding of cyanide (K(d) approximately 0.4 mM) to the heme. In contrast, neither these same variants in the Fe(III) form nor wild-type CooA in either oxidation state was able to bind cyanide even at high concentrations (50 mM). Examination of the pH dependence of spectral changes upon addition of cyanide suggested that the cyanide anion coordinated the heme iron. In addition, the UV-visible absorption spectrum of H77Y Fe(II) CooA without added effectors is also pH-dependent, suggesting that an ionizable amino acid has become solvent-accessible in the absence of His(77). Finally, we demonstrate that the transcriptional activity of H77Y CooA shows a small (1.4-fold) increase in the presence of cyanide, suggesting that the binding of cyanide to this variant promotes the active conformation of H77Y CooA.

Solution 1H NMR study of the heme cavity and folding topology of the abbreviated chain 118-residue globin from the cyanobacterium Nostoc commune.

The globin from the cyanobacterium Nostoc commune, abbreviated GlbN, which appears to serve as a part of a terminal oxidase rather than as a respiratory pigment, displays relatively normal O2 binding properties, despite the highly abbreviated polypeptide chain, (118 residues) relative to more conventional globins [Thorsteinsson, M. V. , Bevan, D. R., Potts, M., Dou, Y., Eich, R. F., Hargrove, M. S., Gibson, Q. H., and Olson, J. S. (1999) Biochemistry 38, 2117-2126]. The nature of the heme cavity and the general folding topology of this cyanoglobin were investigated by solution 1H NMR to establish the extent to which, and the manner in which, this compact globin adheres to the standard globin fold. This represents by far the smallest globin subjected to structural analysis. The paramagnetic cyanomet derivative was selected because its characteristically large magnetic anisotropy imparts significant dipolar shifts which both improve resolution to greatly facilitate assignments and serve as indicators of the folding topology of the globin. Identification of the axial His 70 and highly conserved Phe 35 (CD1) determined the absolute orientation of the heme and proximal His. Sequential assignments of four helical and one loop segments, which exhibit dipolar contacts to the heme and among each other, confirm the presence of well-conserved F, G, and H helices and the FG corner. The majority of the abbreviation of the chain relative to the more conventional length globins is accommodated in the A-D helices, of which the last is completely missing. The distal residue which provides a H-bond to bound ligand is identified as Gln 43, but the expected helical position E7 could not be confirmed. His 46, placed at position E10, is found to adopt alternate orientations into, and out of, the heme cavity depending on protonation state, suggesting the presence of a Bohr effect at low pH. It is shown that the dipolar shifts exhibited by backbone protons for the assigned residues conform well to those observed for other cyanomet globins and further support a conserved Mb fold. Perturbed medium-range dipolar contacts and the pH-independent backbone proton lability of the F helix are interpreted in terms of a holoprotein which is less stable than a conventional length globin.

Electronic absorption, EPR, and resonance raman spectroscopy of CooA, a CO-sensing transcription activator from R. rubrum, reveals a five-coordinate NO-heme.

Electronic absorption, EPR, and resonance Raman spectroscopies revealed that CooA, the CO-sensing transcriptional regulator from Rhodospirillum rubrum, reacts with NO to form a five-coordinate NO-heme. NO must therefore displace both of the heme ligands from six-coordinate, low-spin Fe(II)CooA in forming five-coordinate Fe(II)CooA(NO). CO, in contrast, displaces a single heme ligand from Fe(II)CooA to form six-coordinate Fe(II)CooA(CO). Of a series of common heme-binding ligands, only CO and NO were able to bind to the heme of wild-type CooA; imidazole, azide anion, and cyanide anion had no effect on the heme absorption spectrum. Although NO binds to the heme and displaces the endogenous ligands, NO was not able to induce CooA to bind to its target DNA. The mechanism of CO-dependent activation of CooA is thus more complex than simple displacement of a ligand from the heme iron since NO does not trigger DNA binding. These observations suggest that the CooA heme site discriminates between NO and the biologically relevant signal, CO.

Probing the heme axial ligation in the CO-sensing CooA protein with magnetic circular dichroism spectroscopy.

The combination of UV/visible/near-IR variable-temperature magnetic circular dichroism (VTMCD) and EPR spectroscopies has been used to investigate the spin states and axial ligation of the heme group in oxidized, reduced, and CO-bound reduced forms of the Rhodospirillum rubrum CO oxidation transcriptional activator protein (CooA) and its H77Y and C75S variants. The energy of the porphyrin(pi)-to-Fe(III) charge-transfer band (8930 cm(-)(1)) and the presence of cysteinate S-to-Fe(III) charge-transfer bands between 600 and 700 nm confirm cysteinate axial ligation to the low-spin Fe(III) hemes in oxidized wild-type and H77Y CooA. In contrast, the major component in the oxidized C75S variant is shown to be a low-spin Fe(III) heme with bis-histidine or histidine/amine axial ligation on the basis of the energy of the porphyrin(pi)-to-Fe(III) charge-transfer band (6240 cm(-)(1)) and the anisotropy of the EPR signal, g = 3.23, approximately 2.06, approximately 1.14. These results confirm Cys75 as the cysteinyl axial ligand in oxidized CooA, indicate that it is replaced as an axial ligand by a histidine in the C75S variant, and reveal the presence of a hitherto unidentified histidine or neutral nitrogen ligand trans to Cys75 in wild-type CooA. Evidence for a Cys75-to-His77 axial ligand switch on reduction of CooA comes from VTMCD studies of the reduced proteins. The VTMCD spectra of reduced wild-type and C75S CooA are dominated by bands characteristic of bis-histidine low-spin Fe(II) hemes, whereas the reduced H77Y variant is predominantly high-spin with MCD characteristics typical of a five-coordinate, histidine-ligated ferrous heme. VTMCD studies show that the CO-bound reduced forms of wild-type, H77Y, and C75S contain low-spin Fe(II) hemes and that the Fe-CO bonds can be photolytically cleaved at temperatures <50 K. Strong evidence that CO binding to the heme group in reduced CooA occurs with displacement of His77 comes from the VTMCD spectra of the low-temperature photoproducts of CO-bound reduced forms of wild-type, H77Y, and C75S CooA. The spectra are almost identical to each other and closely correspond to those of the low-temperature photoproducts of well characterized CO-bound ferrous hemes with His/CO axial ligation.

Identification of two important heme site residues (cysteine 75 and histidine 77) in CooA, the CO-sensing transcription factor of Rhodospirillum rubrum.

The CO-sensing mechanism of the transcription factor CooA from Rhodospirillum rubrum was studied through a systematic mutational analysis of potential heme ligands. Previous electron paramagnetic resonance (EPR) spectroscopic studies on wild-type CooA suggested that oxidized (FeIII) CooA contains a low-spin heme with a thiolate ligand, presumably a cysteine, bound to its heme iron. In the present report, electronic absorption and EPR analysis of various substitutions at Cys residues establish that Cys75 is a heme ligand in FeIII CooA. However, characterization of heme stability and electronic properties of purified C75S CooA suggest that Cys75 is not a ligand in FeII CooA. Mutational analysis of all CooA His residues showed that His77 is critical for CO-stimulated transcription. On the basis of findings that H77Y CooA is perturbed in its FeII electronic properties and is unable to bind DNA in a site-specific manner in response to CO, His77 appears to be an axial ligand to FeII CooA. These results imply a ligand switch from Cys75 to His77 upon reduction of CooA. In addition, an interaction has been identified between Cys75 and His77 in FeIII CooA that may be involved in the CO-sensing mechanism. Finally, His77 is necessary for the proper conformational change of CooA upon CO binding.

Resonance Raman evidence for a novel charge relay activation mechanism of the CO-dependent heme protein transcription factor CooA.

Resonance Raman spectra of the CO-responsive transcription factor CooA from Rhodospirillum rubrum provides evidence on the nature of heme ligation and its CO activation mechanism. The Fe(III) form gives standard low-spin heme spectrum, while the Fe(II) form is low spin for wild-type (WT) CooA and mixed spin for a CooA variant, H77Y, with an His77Tyr substitution. The Fe(II) porphyrin skeletal mode nu11 is at a value (1541 cm-1) indicative of a neutral donor ligand for the H77Y variant but is at an unusually depressed frequency (1529 cm-1) for the WT protein, indicating a strongly donating ligand. This ligand is proposed to be His77 imidazolate, formed by proton transfer to a nearby acceptor. The WT CO adduct has FeCO and CO stretching frequencies that indicate a neutral trans ligand and negative polarity in the CO binding pocket, while the CO adduct of His77Tyr has both 6- and 5-coordinate signals and a nonpolar CO environment. Photolysis of the WT CO adduct by the Raman laser produced a low-spin product at steady state, indicating fast recombination of the displaced ligand. These data suggest a novel YH- - -His- charge relay mechanism for CooA activation by CO. In this mechanism, His77 is reprotonated upon ligand displacement by CO; CO displaces either His77 or the trans ligand, X. The resulting charge on Y- may induce the protein conformation change required for site-selective DNA binding.

A cyanobacterial hemoglobin with unusual ligand binding kinetics and stability properties.

The glbN gene of the cyanobacterium Nostoc commune UTEX 584 encodes a hemoprotein, named cyanoglobin, that has high oxygen affinity. The basis for the high oxygen affinity of cyanoglobin was investigated through kinetic studies that utilized stopped-flow spectrophotometry and flash photolysis. Association and dissociation rate constants were measured at 20 degrees C for oxygen, carbon monoxide, nitric oxide, and methyl and ethyl isocyanides. The association rate constants for the binding of these five ligands to cyanoglobin are the highest reported for any naturally occurring hemoglobin, suggesting an unhindered and apolar ligand binding pocket. Cyanoglobin also shows high rates of autoxidation and hemin loss, indicating that the prosthetic group is readily accessible to solvent. The ligand binding behavior of cyanoglobin was more similar to that of leghemoglobin a than to that of sperm whale myoglobin. Collectively, the data support the model of cyanoglobin function described by Hill et al. [(1996) J. Bacteriol. 178, 6587-6598], in which cyanoglobin sequesters oxygen, and presents it to, or is a part of, a terminal cytochrome oxidase complex in Nostoc commune UTEX 584 under microaerobic conditions, when nitrogen fixation, and thus ATP demand, is maximal.

GlbN (cyanoglobin) is a peripheral membrane protein that is restricted to certain Nostoc spp.

The glbN gene of Nostoc commune UTEX 584 is juxtaposed to nifU and nifH, and it encodes a 12-kDa monomeric hemoglobin that binds oxygen with high affinity. In N. commune UTEX 584, maximum accumulation of GlbN occurred in both the heterocysts and vegetative cells of nitrogen-fixing cultures when the rate of oxygen evolution was repressed to less than 25 micromol of O2 mg of chlorophyll a(-1) h(-1). Accumulation of GlbN coincided with maximum synthesis of NifH and ferredoxin NADP+ oxidoreductase (PetH or FNR). A total of 41 strains of cyanobacteria, including 40 nitrogen fixers and representing 16 genera within all five sections of the cyanobacteria were screened for the presence of glbN or GlbN. glbN was present in five Nostoc strains in a single copy. Genomic DNAs from 11 other Nostoc and Anabaena strains, including Anabaena sp. strain PCC 7120, provided no hybridization signals with a glbN probe. A constitutively expressed, 18-kDa protein which cross-reacted strongly with GlbN antibodies was detected in four Anabaena and Nostoc strains and in Trichodesmium thiebautii. The nifU-nifH intergenic region of Nostoc sp. strain MUN 8820 was sequenced (1,229 bp) and was approximately 95% identical to the equivalent region in N. commune UTEX 584. Each strand of the DNA from the nifU-nifH intergenic regions of both strains has the potential to fold into secondary structures in which more than 50% of the bases are internally paired. Mobility shift assays confirmed that NtcA (BifA) bound a site in the nifU-glbN intergenic region of N. commune UTEX 584 approximately 100 bases upstream from the translation initiation site of glbN. This site showed extensive sequence similarity with the promoter region of glnA from Synechococcus sp. strain PCC 7942. In vivo, GlbN had a specific and prominent subcellular location around the periphery of the cytosolic face of the cell membrane, and the protein was found solely in the soluble fraction of cell extracts. Our hypothesis is that GlbN scavenges oxygen for and is a component of a membrane-associated microaerobically induced terminal cytochrome oxidase.

Spectroscopical and functional characterization of the hemoglobin of Nostoc commune (UTEX 584 (Cyanobacterial).

Structural analysis of a monomeric hemoglobin from the cyanobacterium Nostoc commune strain UTEX 584, cyanoglobin (Potts et al. (1992) Science 256, 1690-1692), is presented. Cyanoglobin binds molecular oxygen reversibly, with high oxygen affinity and non-cooperativity. There was no evidence for decreased stability of the pigment at 37 degrees C. Cyanoglobin-specific antibodies showed no cross-reactivity with two reference hemoglobins, leghemoglobin a and sperm whale myoglobin. The absorption spectral properties of cyanoglobin differ significantly from those of the two reference hemoglobins. The spectrum of oxy-cyanoglobin most closely resembles that of an oxy-hemoglobin from the protozoan Tetrahymena pyriformis, a hemoprotein that shares substantial amino-acid sequence identity with cyanoglobin. Met-cyanoglobin possesses spectral characteristics at pH 7.0-9.0 that resemble those of the alkaline met-hemoglobin (a putative hemichrome) of another protozoan, Paramecium caudatum. The spin-state character of met-cyanoglobin is pH-dependent. Met-cyanoglobin does not coordinate the strong-field ligands, cyanide and azide, at pH 7.0. The capacity of cyanoglobin to coordinate cyanide increased with decreasing pH. Far-UV CD spectra of cyanoglobin are indicative of a protein with a significant amount of alpha-helical structure. Data from Soret-region CD spectra suggest that the orientations of the heme moieties in cyanoglobin and leghemoglobin a are similar to one another.

Inhibition of an archaeal protein phosphatase activity by okadaic acid, microcystin-LR, or calyculin A.

Soluble extracts of the methanogenic archaeon, Methanosarcina thermophila TM-1, contained a divalent metal ion-stimulated protein-serine phosphatase activity. This activity was sensitive to micromolar concentrations of okadaic acid, microcystin-LR, or calyculin A, three compounds thought to be highly specific inhibitors of the type 1/2A/2B genetic superfamily of eukaryotic protein-serine/threonine phosphatases. The observation that each of these three chemically unrelated compounds inhibited this archaeal protein phosphatase activity suggests the existence of structural homology, and perhaps even common genetic ancestry, with the type 1/2A/2B superfamily of protein-serine/threonine phosphatases found in eukaryotic organisms.