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• W2053682017 abstract "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. Gly117 is one of the residues on the C-helix within 7 Å of the heme iron on the Pro2 side of the heme in CooA. Analysis of a series of Gly117 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 Gly117 variant, G117I CooA, was specifically perturbed in its Pro2 ligation in both Fe(III) and Fe(II) forms, but comparison with other CooA variants indicated that perturbation of Pro2 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 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. Gly117 is one of the residues on the C-helix within 7 Å of the heme iron on the Pro2 side of the heme in CooA. Analysis of a series of Gly117 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 Gly117 variant, G117I CooA, was specifically perturbed in its Pro2 ligation in both Fe(III) and Fe(II) forms, but comparison with other CooA variants indicated that perturbation of Pro2 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. electron paramagnetic resonance cAMP receptor protein fumarate and nitrate reductase activator protein wild-type 4-morpholinepropanesulfonic acid cytochrome P-450 cam dithiothreitol cyanide anion Proteins that sense small gaseous molecules, such as NO, O2, and CO, play important roles in biological systems ranging from prokaryotes to mammals. These include soluble guanylyl cyclase, which binds NO and exerts a variety of physiological responses, including neurotransmission and smooth muscle vasodilation (1Bredt D.S. Snyder S.H. Annu. Rev. Biochem. 1994; 63: 175-195Crossref PubMed Scopus (2147) Google Scholar, 2Ignarro L.J. Biochem. Pharmacol. 1991; 41: 485-490Crossref PubMed Scopus (491) Google Scholar, 3Marletta M.A. Cell. 1994; 78: 927-930Abstract Full Text PDF PubMed Scopus (817) Google Scholar), and FixL, which binds O2 and regulates gene expression in nitrogen-fixing rhizobia (4Gilles-Gonzales M.A. Ditta G.S. Helinski D.R. Nature. 1991; 350: 170-172Crossref PubMed Scopus (417) Google Scholar, 5Lukat-Rodgers G.S. Rodgers K.R. Biochemistry. 1997; 36: 4178-4187Crossref PubMed Scopus (79) Google Scholar). The only CO-sensing protein described thus far, CooA, was found in the photosynthetic bacterium Rhodospirillum rubrum, which can utilize CO as a sole energy source (6Kerby R.L. Ludden P.W. Roberts G.P. J. Bacteriol. 1995; 177: 2241-2244Crossref PubMed Google Scholar). CooA uses a b-type heme to bind CO, whereupon the protein undergoes a conformational change that allows it to bind DNA and activate the transcription of genes encoding the CO-oxidation system (7Shelver D. Thorsteinsson M.V. Kerby R.L. Chung S.Y. Roberts G.P. Reynolds M.F. Parks R.B. Burstyn J.N. Biochemistry. 1999; 38: 2669-2678Crossref PubMed Scopus (100) Google Scholar). CooA is a homodimeric protein containing an effector-binding domain, which contains the heme, and a DNA-binding domain linked by a hinge region. The heme of CooA is six-coordinate and low spin in its Fe(III), Fe(II), and Fe(II)-CO forms, implying that CO must displace one of the axial ligands of Fe(II) CooA. Recently, the x-ray crystal structure of Fe(II) CooA was solved to 2.6-Å resolution and revealed that Pro2 of one monomer serves as one ligand to the heme iron of the other monomer and that the ligand trans to Pro2 is His77 (8Lanzilotta W.N. Schuller D.J. Thorsteinsson M.V. Kerby R.L. Roberts G.P. Poulos T.L. Nat. Struct. Biol. 2000; 7: 876-880Crossref PubMed Scopus (235) Google Scholar). Previous studies, including electron paramagnetic resonance (EPR)1analysis and site-directed mutagenesis showed that one of the ligands in the Fe(III) form is Cys75 in the thiolate form (7Shelver D. Thorsteinsson M.V. Kerby R.L. Chung S.Y. Roberts G.P. Reynolds M.F. Parks R.B. Burstyn J.N. Biochemistry. 1999; 38: 2669-2678Crossref PubMed Scopus (100) Google Scholar, 9Reynolds M.F. Shelver D. Kerby R.L. Parks R.B. Roberts G.P. Burstyn J.N. J. Am. Chem. Soc. 1998; 120: 9080-9081Crossref Scopus (56) Google Scholar), and Pro2 is assumed to be the trans ligand, based on indirect evidence (10Thorsteinsson M.V. Kerby R.L. Conrad M. Youn H. Staples C.R. Lanzilotta W.N. Poulos T.J. Serate J. Roberts G.P. J. Biol. Chem. 2000; 275: 39332-39338Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 11Nakajima H. Honma Y. Tawara T. Kato T. Park S.-Y. Miyatake H. Shiro Y. Aono S. J. Biol. Chem. 2001; 276: 7055-7061Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Thus, there is an unusual ligand switch upon reduction of the heme, with His77 replacing Cys75 (7Shelver D. Thorsteinsson M.V. Kerby R.L. Chung S.Y. Roberts G.P. Reynolds M.F. Parks R.B. Burstyn J.N. Biochemistry. 1999; 38: 2669-2678Crossref PubMed Scopus (100) Google Scholar, 12Aono S. Ohkubo K. Matsuo T. Nakajima H. J. Biol. Chem. 1998; 273: 25757-25764Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). CO binding has been shown to displace Pro2 (10Thorsteinsson M.V. Kerby R.L. Conrad M. Youn H. Staples C.R. Lanzilotta W.N. Poulos T.J. Serate J. Roberts G.P. J. Biol. Chem. 2000; 275: 39332-39338Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 11Nakajima H. Honma Y. Tawara T. Kato T. Park S.-Y. Miyatake H. Shiro Y. Aono S. J. Biol. Chem. 2001; 276: 7055-7061Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 13Yamamoto K. Ishikawa H. Takahashi S. Ishimori K. Morishima I. Nakajima H. Aono S. J. Biol. Chem. 2001; 276: 11473-11476Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), but a variety of Pro2variants turned out to be CO-responsive, indicating that Pro2 is not critical for activation of CooA in response to CO (10Thorsteinsson M.V. Kerby R.L. Conrad M. Youn H. Staples C.R. Lanzilotta W.N. Poulos T.J. Serate J. Roberts G.P. J. Biol. Chem. 2000; 275: 39332-39338Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Cys75 is also not essential for activity, because C75S CooA shows CO-dependent activity, although the heme is unstable in the Fe(III) form (7Shelver D. Thorsteinsson M.V. Kerby R.L. Chung S.Y. Roberts G.P. Reynolds M.F. Parks R.B. Burstyn J.N. Biochemistry. 1999; 38: 2669-2678Crossref PubMed Scopus (100) Google Scholar). The nature of the residue at position 77 is, however, absolutely critical for CO-dependent conformational changes of CooA (7Shelver D. Thorsteinsson M.V. Kerby R.L. Chung S.Y. Roberts G.P. Reynolds M.F. Parks R.B. Burstyn J.N. Biochemistry. 1999; 38: 2669-2678Crossref PubMed Scopus (100) Google Scholar). CooA variants altered at this position still bind CO but fail to be activated. In at least one such variant, CO is apparently bound to the “wrong” side of the heme (13Yamamoto K. Ishikawa H. Takahashi S. Ishimori K. Morishima I. Nakajima H. Aono S. J. Biol. Chem. 2001; 276: 11473-11476Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), which might be the basis for the lack of CO activation. CooA is a member of the cAMP receptor protein (CRP)/fumarate and nitrate reductase activator protein (FNR) family of transcriptional regulators (14Shelver D. Kerby R.L. He Y. Roberts G.P. J. Bacteriol. 1995; 177: 2157-2163Crossref PubMed Google Scholar). These proteins exert their function by binding a cognate DNA sequence after responding to their specific effectors (cAMP for CRP; reducing conditions for FNR; CO for CooA). The only structure known for CRP is the cAMP-bound form (15Passner J.M. Steitz T.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2843-2847Crossref PubMed Scopus (161) Google Scholar), and a variety of analyses have suggested that a substantial conformational change occurs upon effector binding (16Won H.-S. Yamazaki T. Lee T.-W. Yoon M.-K. Park S.-H. Kyogoku Y. Lee B.-J. Biochemistry. 2000; 39: 13953-13962Crossref PubMed Scopus (59) Google Scholar, 17Baichoo N. Heyduk T. Biochemistry. 1997; 36: 10830-10836Crossref PubMed Scopus (46) Google Scholar, 18Cheng X. Kovac L. Lee J.C. Biochemistry. 1995; 34: 10816-10826Crossref PubMed Scopus (38) Google Scholar). No structure has been obtained for FNR, although it appears to be activated by a very different mechanism of a monomer-dimer transition rather than a conformational change within a dimer, as in the case of CRP and CooA (19Lazazzera B.A. Beinert H. Khoroshilova N. Kennedy M.C. Kiley P.J. J. Biol. Chem. 1996; 271: 2762-2768Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). The structural comparison of Fe(II) CooA in the absence of effector (CO) with CRP bound to its effector (cAMP) suggested a number of differences between them, among which was a modest repositioning of the long C-helices that serve as the dimer interface in both CRP and CooA (8Lanzilotta W.N. Schuller D.J. Thorsteinsson M.V. Kerby R.L. Roberts G.P. Poulos T.L. Nat. Struct. Biol. 2000; 7: 876-880Crossref PubMed Scopus (235) Google Scholar). This has lead to the hypothesis that in CooA, effector binding causes a change in the relative positions of the C-helices, which transmits a signal to the DNA-binding domains. Presumably this repositioning either destabilizes the inactive conformation, stabilizes the active conformation, or both. It is unclear how CO binding might affect the repositioning of the C-helix, although it seems plausible that CO displacement of Pro2 allows the heme, still tethered to His77, to interact with the C-helices. To test this hypothesis, we examined the surface of the C-helices in the structure of Fe(II) CooA and noted that there are relatively few residues that are in a position to interact with the heme (8Lanzilotta W.N. Schuller D.J. Thorsteinsson M.V. Kerby R.L. Roberts G.P. Poulos T.L. Nat. Struct. Biol. 2000; 7: 876-880Crossref PubMed Scopus (235) Google Scholar): Gly117, Leu112, Ile113, and Leu116. In this paper, we report the importance of the heme pocket affected by substitution at the Gly117 position and the analysis provides a number of insights into the response of wild-type (WT) CooA to CO. WT CooA and CooA variants were constructed in an Escherichia coli overexpression system and a β-galactosidase reporter system as described previously (7Shelver D. Thorsteinsson M.V. Kerby R.L. Chung S.Y. Roberts G.P. Reynolds M.F. Parks R.B. Burstyn J.N. Biochemistry. 1999; 38: 2669-2678Crossref PubMed Scopus (100) Google Scholar). In vivoβ-galactosidase activity in an E. coli reporter system was monitored as described previously (7Shelver D. Thorsteinsson M.V. Kerby R.L. Chung S.Y. Roberts G.P. Reynolds M.F. Parks R.B. Burstyn J.N. Biochemistry. 1999; 38: 2669-2678Crossref PubMed Scopus (100) Google Scholar) and quantitated using the standard protocol (20Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Short Protocols in Molecular Biology.in: 3rd Ed. John Wiley & Sons, Inc., New York1995: 13-30Google Scholar). The purification of WT CooA and CooA variants to ∼95% purity was performed as described previously (7Shelver D. Thorsteinsson M.V. Kerby R.L. Chung S.Y. Roberts G.P. Reynolds M.F. Parks R.B. Burstyn J.N. Biochemistry. 1999; 38: 2669-2678Crossref PubMed Scopus (100) Google Scholar). The heme content of CooA preparations was quantitated by the reduced pyridine-hemochromogen method (21DeDuve C. Acta Chem. Scand. 1948; 2: 264-289Crossref PubMed Google Scholar). For the initial screening of the heme coordination states of different CooA variants, a 50-ml culture of the cells was harvested, resuspended in 5 ml of 25 mm MOPS buffer, 0.2 mNaCl, 10% glycerol, pH 7.4, broken with a French pressure cell (∼120 MPa), and centrifuged for 30 min at 11,700 × g. The supernatant was then mixed with 0.3 g of solid hydroxyapatite resin. After unbound materials were removed from the resin, a high salt buffer containing 25 mm MOPS, pH 7.4, 10 mmpotassium phosphate, pH 7.4, 1.2 m KCl, and 5% glycerol was added, and the resin was washed twice. CooA was then eluted with high phosphate buffer containing 25 mm MOPS, pH 7.4, 160 mm potassium phosphate, pH 7.4, 50 mm KCl, and 5% glycerol. The eluent was precipitated with ammonium sulfate with a final saturation of 50% and stored at −20 °C until use. CooA prepared with this procedure resulted in an enrichment of CooA to ∼10% of total protein. Electronic absorption spectra of CooA variants were measured with a Shimadzu UV-2401 spectrophotometer. Anaerobic reduction of CooA samples and anaerobic addition of endogenous ligands such as CO, KCN, and imidazole were carried out as described previously (7Shelver D. Thorsteinsson M.V. Kerby R.L. Chung S.Y. Roberts G.P. Reynolds M.F. Parks R.B. Burstyn J.N. Biochemistry. 1999; 38: 2669-2678Crossref PubMed Scopus (100) Google Scholar, 22Shelver D. Kerby R.L. He Y. Roberts G.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11216-11220Crossref PubMed Scopus (192) Google Scholar, 23Thorsteinsson M.V. Kerby R.L. Roberts G.P. Biochemistry. 2000; 39: 8284-8290Crossref PubMed Scopus (23) Google Scholar). EPR spectra were recorded on a Varian E-15 spectrometer with an Oxford Cryostat 3120 system to regulate and monitor the temperature at a microwave frequency of 9.25 GHz at 4 K and 200 microwatts (μW) for high spin signals and 22 K and 20 μW for low spin signals. EPR spectra at pH 7.4 were obtained in 25 mm MOPS buffer, pH 7.4, containing 0.1 m NaCl. For the preparation of Fe(III) G117I CooA at pH 6.6 and 9.4 for EPR, samples were buffered by mixing 150 μl of the purified protein (dissolved in 25 mm MOPS, pH 7.4, and 0.1 mNaCl) with 150 μl of 200 mm buffer (MOPS, pH 6.5; glycine/NaOH, pH 9.5) containing 0.1 m NaCl to provide a final buffer concentration of ∼100 mm. EPR samples of Fe(III) G117I CooA and WT CooA at pH 3.4 were buffered by mixing 150 μl of the purified protein (dissolved in 25 mm MOPS, pH 7.4, and 0.1 m NaCl) with 150 μl of 400 mmglycine/HCl, pH 3.1, and 0.1 m NaCl to provide a final buffer concentration of ∼200 mm. For the EPR analysis of ΔP3R4 CooA at pH 10.2, isolated Fe(III) ΔP3R4 CooA was buffered by using 200 mm glycine/NaOH, pH 10.5, and 0.1 mNaCl. The pH of all samples for EPR spectra was confirmed after mixing using an Orion model 611 pH meter equipped with a Ross semi-micro temperature compensation electrode. The heme concentrations of WT, G117I, and ΔP3R4 CooA for EPR analyses were 145, 82, and 185 μm, respectively. Finally, all the G117I CooA samples used for UV-visible and EPR spectra contained 5 mmdithiothreitol (DTT) as a stabilizing agent. The effect of pH on the UV-visible spectrum of WT CooA was monitored by adding 10 μl of 610 μm WT CooA to 990 μl of different buffer solutions. The following buffer solutions were used: pH 7.4, 100 mm MOPS, 0.1 m NaCl; pH 6.9, 100 mm MOPS, 0.1m NaCl; pH 6.1, 100 mm potassium phosphate, 0.1m NaCl; pH 5.5, 5.3, 5.0, 4.5, 4.0, 100 mmacetate, 0.1 m NaCl; pH 3.4, 100 mmglycine/HCl, 0.1 m NaCl. In vitro DNA binding assays were performed using the fluorescence polarization assay described previously (10Thorsteinsson M.V. Kerby R.L. Conrad M. Youn H. Staples C.R. Lanzilotta W.N. Poulos T.J. Serate J. Roberts G.P. J. Biol. Chem. 2000; 275: 39332-39338Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Target DNA containing pCooF (purchased from Genosys) was labeled with the fluorescent dye, Texas Red, on one end of the duplex and used at the concentration of 6.4 nm. Dissociation constants (Kd) were calculated by fitting the binding data to a non-linear fitting equation described by Lundblad et al. (24Lundblad J.R. Laurance M. Goodman R.H. Mol. Endocrinol. 1996; 10: 607-612Crossref PubMed Scopus (217) Google Scholar). The crystal structure of Fe(II) CooA revealed that relatively few residues are present in the vicinity of the heme on the side of Pro2, one of the axial ligands in Fe(II) CooA (8Lanzilotta W.N. Schuller D.J. Thorsteinsson M.V. Kerby R.L. Roberts G.P. Poulos T.L. Nat. Struct. Biol. 2000; 7: 876-880Crossref PubMed Scopus (235) Google Scholar). These residues include Gly117, and we tested the hypothesis that the portion of the pocket provided by this residue might be important for heme interaction with the C-helix upon CO binding (Fig.1). The set of Gly117variants studied here included G117A, G117S, G117V, G117I, and G117H. As shown in Table I, the activities of these Gly117 variants in response to CO are severely perturbed in our in vivo assay. As the size of substitution at the position 117 increased (see “mole vol ” column in Table I), CO-sensing function was reduced. This CO-sensing function is indicated in Table I by the ratio of the activity of each CooA variant in the presence of CO to that in the absence of CO; this ratio is an approximate measure of the -fold activation caused by effector binding. There is also a less striking correlation between residue size and effector-independent activity: Larger residues at position 117 provided greater effector-independent activity in the Fe(III) and Fe(II) forms (Table I). It is important to note that the differences in activity in vivo of the variants are not the result of poor accumulation of heme-containing CooA, because we have shown that ∼5% active CooA (compared with WT CooA) is sufficient for maximal in vivo activity (25Thorsteinsson M.V. Kerby R.L. Youn H. Conrad M. Serate J. Staples C.R. Roberts G.P. J. Biol. Chem. 2001; 276: 26807-26813Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar) and all the Gly117 variants accumulate ≥25% of WT CooA (Table I).Table IProperties of CooA variants altered in the vicinity of heme through site-directed mutagenesisCooASoret maximumSoret peak ratioβ-galactosidase activitya% activity indicates the mean value of multiple measurements of activity relative to that of WT CooA in the presence of CO, and showed variability <10%.Mole VoleMolecular volume (35).Accumulationb% accumulation indicates the heme accumulation of variant CooA in cell-free extracts variant relative to that of WT CooA, based on the spectra of CO-bound form.Fe(III)Fe(II)Fe(II)-COAFe(II)/AFe(III)AFe(II)-CO/AFe(III)OxicAerobically grown cells were used for the activity.ReddAnaerobically grown cells were used for the activity.−CORed + CORatio (+CO/−CO)%Å3%pKK223–30.50.510WTfPurified sample was used for measurement of Soret maximum.423.5424.54222.07gFor comparison,AFe(III) for WT CooA was obtained from the UV-visible spectrum at pH 3.4 and AFe(II) and AFe(II)-CO for WT CooA were obtained from those at pH 7.4. The same amount of WT CooA was used for the UV-visible spectra at both pH 3.4 and 7.4.2.17gFor comparison,AFe(III) for WT CooA was obtained from the UV-visible spectrum at pH 3.4 and AFe(II) and AFe(II)-CO for WT CooA were obtained from those at pH 7.4. The same amount of WT CooA was used for the UV-visible spectra at both pH 3.4 and 7.4.2310033.360.1100G117AfPurified sample was used for measurement of Soret maximum.WThWT indicates the same Soret maximum and peak shape as WT CooA.WTWT233411.388.637G117SiHydroxyapatite batch-enriched CooA sample was used for Soret maximum.WTWTWT46193.289.054G117ViHydroxyapatite batch-enriched CooA sample was used for Soret maximum.387425j↓ indicates reduced intensity in Soret band compared to WT CooA.↓WT913161.2140.030G117HfPurified sample was used for measurement of Soret maximum.WTWTWT61090.9153.225G117IfPurified sample was used for measurement of Soret maximum.387426 ↓WT1.172.0861160.5166.735ΔP3-I7iHydroxyapatite batch-enriched CooA sample was used for Soret maximum.387425 ↓WT22705ΔP3R4fPurified sample was used for measurement of Soret maximum.387425 ↓WT1.682.062713110G117A ΔP3R4iHydroxyapatite batch-enriched CooA sample was used for Soret maximum.387425 ↓WT131628G117H ΔP3R4iHydroxyapatite batch-enriched CooA sample was used for Soret maximum.WTWTWT35525G117I ΔP3R4fPurified sample was used for measurement of Soret maximum.387426 ↓WT1.152.0822225G117I C75ANDkND, not detectable.NDND244<1G117I H77AfPurified sample was used for measurement of Soret maximum.387417 ↓4190.821.691011825a % activity indicates the mean value of multiple measurements of activity relative to that of WT CooA in the presence of CO, and showed variability <10%.b % accumulation indicates the heme accumulation of variant CooA in cell-free extracts variant relative to that of WT CooA, based on the spectra of CO-bound form.c Aerobically grown cells were used for the activity.d Anaerobically grown cells were used for the activity.e Molecular volume (35Zamyatin A.A. Prog. Biophys. Mol. Biol. 1972; 24: 107-123Crossref PubMed Scopus (449) Google Scholar).f Purified sample was used for measurement of Soret maximum.g For comparison,AFe(III) for WT CooA was obtained from the UV-visible spectrum at pH 3.4 and AFe(II) and AFe(II)-CO for WT CooA were obtained from those at pH 7.4. The same amount of WT CooA was used for the UV-visible spectra at both pH 3.4 and 7.4.h WT indicates the same Soret maximum and peak shape as WT CooA.i Hydroxyapatite batch-enriched CooA sample was used for Soret maximum.j ↓ indicates reduced intensity in Soret band compared to WT CooA.k ND, not detectable. Open table in a new tab The Soret wavelength maxima of Gly117 variants are also listed in Table I. Although substitutions at position 117 with relatively smaller side chains such as Ala and Ser showed wavelength maxima that were indistinguishable from that of WT CooA, G117V CooA and G117I CooA revealed a blue-shifted Soret peak at 387 nm in the Fe(III) form, characteristic of a five-coordinate high spin heme. The Fe(II) forms of G117V CooA and G117I CooA are also severely perturbed in terms of Soret maxima, and the detailed spectral properties of purified G117I CooA will be discussed below. The extent of the perturbation of both UV-visible spectra and the CO-sensing function in Gly117variants was well correlated with the size of the substitutions (TableI), with the exception of G117H, which will be discussed below. This result indicates that the portion of the heme pocket created by Gly117 is important for the ligation structure and CO-sensing function of CooA. As shown in Fig.2 B, the UV-visible spectrum of purified Fe(III) G117I CooA is typical for a thiolate-ligated five-coordinate high spin heme with a Soret peak at 387 nm and ligand-to-metal charge-transfer peak appearing at ∼640 nm. The EPR spectrum of Fe(III) G117I CooA corroborated the results of the UV-visible spectrum, displaying only high spin signal with a single population corresponding to gx = 7.15,gy = 5.02, and gz = 1.93 (Fig. 3 and TableII). This result indicates that one of the two axial ligands in the six-coordinate Fe(III) WT CooA (Fig.2 A) is selectively perturbed in the Fe(III) G117I CooA variant. The rhombicity (expressed as E/D (26Tsai A.-L. Berka V. Chen P.-F. Palmer G. J. Biol. Chem. 1996; 271: 32563-32571Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar)) of G117I CooA is comparable to that of a model heme-thiolate complex (Table II (27Tang S.C. Koch S. Papaefthymiou G.C. Foner S. Frankel R.B. Ibers J.A. Holm R.H. J. Am. Chem. Soc. 1976; 98: 2414-2434Crossref PubMed Scopus (254) Google Scholar)), although it is a bit lower than those of Fe(III) five-coordinate, high spin thiolate-ligated hemoproteins such as cytochrome P450 cam (P-450 cam (28Lipscomb J.D. Biochemistry. 1980; 19: 3590-3599Crossref PubMed Scopus (161) Google Scholar)), endothelial nitric oxide synthase (26Tsai A.-L. Berka V. Chen P.-F. Palmer G. J. Biol. Chem. 1996; 271: 32563-32571Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), and chloroperoxidase (29Hollenberg P.F. Hager L.P. Blumberg W.E. Peisach J. J. Biol. Chem. 1980; 255: 4801-4807Abstract Full Text PDF PubMed Google Scholar) (Table II). On the other hand, the rhombicity of G117I CooA is significantly higher than those of nitrogen-based ligand high spin signal such as FixL (30Lukat-Rodgers G.S. Rexine J.L. Rodgers K.R. Biochemistry. 1998; 37: 13543-13552Crossref PubMed Scopus (29) Google Scholar) and soluble guanylyl cyclase (31Stone J.R. Sands R.H. Dunham W.R. Marletta M.A. Biochemistry. 1996; 35: 3258-3262Crossref PubMed Scopus (51) Google Scholar). The results of UV-visible and EPR spectroscopies strongly suggest that the retained ligand in Fe(III) G117I CooA is a thiolate.Figure 3X-band EPR spectra of WT CooA and CooA variants. The samples were prepared as described under “Experimental Procedures”; in case of G117I CooA samples 5 mm DTT was included for increased stability. The EPR conditions for the detection of low spin signals were set to 22 K and 20 μW in the case of G117I CooA at pH 9.4 and ΔP3R4 CooA at pH 10.2 (indicated by the asterisk). Data were obtained at 4 K and 200 μW for the detection of high spin signals in all other samples.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IIEPR parameters of selected CooA and other thiolated-ligated heme proteinsProteinsHigh spinLow spinReferencegxgygzE/DaE/D is used as a measure of rhombicity.gzgygxWT CooA (pH 7.4)NDbND, not detectable.NDND2.462.251.8992.582.251.84WT CooA (pH 3.4)7.294.991.920.048NDNDNDThis work6.06.02.00G117I CooA (pH 7.4)7.155.021.930.044NDNDNDThis workΔP3R4 CooA (pH 7.4)7.084.911.93cCalculated value.0.045NDNDNDThis work8.073.61cCalculated value.1.71cCalculated value.0.093ΔP3R4 CooA (pH 10.2)NTdNT, not determined.NTNT2.452.261.90This workFe(PPIXDME)(SC6H4Cl)ePPIXDME = protoporphyrin IX dimethyl ester; SC6H4Cl =p-chlorobenzenethiol.7.24.81.90.05027eNOS7.674.341.870.06926P-450 cam7.853.971.780.08128Chloroperoxidase7.64.31.80.06929FixL6.165.751.990.00930Soluble granylyl cyclase6.365.162.000.02531a E/D is used as a measure of rhombicity.b ND, not detectable.c Calculated value.d NT, not determined.e PPIXDME = protoporphyrin IX dimethyl ester; SC6H4Cl =p-chlorobenzenethiol. Open table in a new tab Because Cys75 is a normal ligand in Fe(III) WT CooA, we tested the hypothesis that the thiolate ligand seen above was due to this residue. We reasoned that altering Cys75 in a G117I background would have a profound effect on the protein if Cys75 was the ligand but should have no effect if another Cys residue served that role. A strain with both G117I and C75A substitutions in CooA accumulated <1% the normal level of CooA, consistent with the hypothesis that Cys75 is the ligand in Fe(III) G117I CooA (Table I). As controls, two other variants were constructed: G117I H77A and G117I ΔP3R4. H77A CooA lacks the normal His ligand of the Fe(II) form of WT CooA, and ΔP3R4 CooA is probably perturbed in the normal Pro2 ligand in both Fe(II) and Fe(III) forms of WT CooA because of the deletion of the two adjacent residues. The UV-visible spectrum of Fe(III) G117I CooA was not significantly altered in the ΔP3R4 (data not shown) or H77A backgrounds (Fig. 2 C), indicating that neither His77 nor Pro2 is likely the retained ligand in Fe(III) G117I CooA. In summary, Fe(III) G117I CooA exists only as a five-coordinate high spin heme with Cys75 as the axial ligand, indicating that Pro2, the ligand transto Cys75, is severely perturbed in Fe(III) G117I CooA. One of the general characteristics of CooA variants such as C75S and P2Y is that these variants display a significant amount of six-coordinate heme under conditions where the altered residues would have served as the normal ligand (7Shelver D. Thorsteinsson M.V. Kerby R.L. Chung S.Y. Roberts G.P. Reynolds M.F. Parks R.B. Burstyn J.N. Biochemistry. 1999; 38: 2669-2678Crossref PubMed Scopus (100) Google Scholar, 10Thorsteinsson M.V. Kerby R.L. Conrad M. Youn H. Staples C.R. Lanzilotta W.N. Poulos T.J. Serate J. Roberts G.P. J. Biol. Chem. 2000; 275: 39332-39338Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). This indicates that there are adventitious ligands that partially substitute for the altered ligands. UV-visible and EPR spectra of Fe(" @default.
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