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• W2079852793 abstract "The heme-containing protein CooA of Rhodospirillum rubrum regulates the expression of genes involved in CO oxidation. CooA binds its target DNA sequence in response to CO binding to its heme. Activity measurements and resonance Raman (RR) spectra are reported for CooA variants that bind DNA even in the absence of CO, those in which the wild-type residues at the 121-126 positions, TSCMRT, are replaced by the residues AYLLRL or RYLLRL, and also for variants that bind DNA poorly in the presence of CO, such as L120S and L120F. The Fe-C and C-O stretching resonance Raman (RR) frequencies of all CooAs examined deviate from the expected back-bonding correlation in a manner indicating weakening of the Fe-His-77 proximal ligand bond, and the extent of weakening correlates positively with DNA binding activity. The (A/R) YLLRL variants have detectable populations of a 5-coordinate heme resulting from partial dissociation of the endogenous distal ligand, Pro-2. Selective excitation of this population reveals downshifted Fe-His-77-stretching RR bands, confirming the proximal bond weakening. These results support our previous hypothesis that the conformational change required for DNA binding is initiated by displacement of the heme into an adjacent hydrophobic cavity once CO displaces the Pro-2 ligand. Examination of the crystal structure reveals a physical basis for these results, and a mechanism is proposed to link heme displacement to conformational change. The heme-containing protein CooA of Rhodospirillum rubrum regulates the expression of genes involved in CO oxidation. CooA binds its target DNA sequence in response to CO binding to its heme. Activity measurements and resonance Raman (RR) spectra are reported for CooA variants that bind DNA even in the absence of CO, those in which the wild-type residues at the 121-126 positions, TSCMRT, are replaced by the residues AYLLRL or RYLLRL, and also for variants that bind DNA poorly in the presence of CO, such as L120S and L120F. The Fe-C and C-O stretching resonance Raman (RR) frequencies of all CooAs examined deviate from the expected back-bonding correlation in a manner indicating weakening of the Fe-His-77 proximal ligand bond, and the extent of weakening correlates positively with DNA binding activity. The (A/R) YLLRL variants have detectable populations of a 5-coordinate heme resulting from partial dissociation of the endogenous distal ligand, Pro-2. Selective excitation of this population reveals downshifted Fe-His-77-stretching RR bands, confirming the proximal bond weakening. These results support our previous hypothesis that the conformational change required for DNA binding is initiated by displacement of the heme into an adjacent hydrophobic cavity once CO displaces the Pro-2 ligand. Examination of the crystal structure reveals a physical basis for these results, and a mechanism is proposed to link heme displacement to conformational change. An increasing volume of research has revealed the ubiquity of heme sensor proteins (1Gilles-Gonzalez M. Gonzalez G. J. Inorg. Biochem. 2005; 99: 1-22Crossref PubMed Scopus (295) Google Scholar, 2Uchida T. Kitagawa T. Acc. Chem. Res. 2005; 38: 662-670Crossref PubMed Scopus (75) Google Scholar), which regulate a range of biological activities in response to changing levels of the gaseous molecules CO, NO, or O2. In these proteins nature has taken advantage of the ability of heme to bind these nonpolar molecules and has coupled the elementary act of ligand binding to a change of protein activity. How this is accomplished has become a central issue in chemical biology. One of the best opportunities to probe this question is presented by the well studied molecule CooA (3Roberts G.P. Kerby R.L. Youn H. Conrad M. J. Inorg. Biochem. 2005; 99: 280-292Crossref PubMed Scopus (82) Google Scholar, 4Aono S. Acc. Chem. Res. 2003; 36: 825-831Crossref PubMed Scopus (91) Google Scholar) from Rhodospirillum rubrum, which grows on CO as the sole energy source under anaerobic conditions. When bound by CO, CooA binds its target DNA sequence and activates transcription of genes coding for proteins that oxidize CO to CO2 and reduce protons to H2. CooA is a homodimer, and each 221-residue monomer contains an N-terminal heme binding domain and a C-terminal DNA binding domain. The x-ray crystal structure has been determined for the CO-free inactive state of Fe(II) CooA (5Lanzilotta 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 (234) Google Scholar) but not for the CO-bound active state. However, CooA is homologous to another well studied transcription factor, cAMP receptor protein (CRP) 4The abbreviations used are: CRP, cAMP receptor protein; RR, resonance Raman; WT, wild-type; MOPS, 4-morpholinepropanesulfonic acid; Mb, myoglobin; 6-c, 6-coordinate. 4The abbreviations used are: CRP, cAMP receptor protein; RR, resonance Raman; WT, wild-type; MOPS, 4-morpholinepropanesulfonic acid; Mb, myoglobin; 6-c, 6-coordinate. (6Weber I.T. Steitz T.A. J. Mol. Biol. 1987; 198: 311-326Crossref PubMed Scopus (408) Google Scholar, 7Passner J.M. Schultz S.C. Steitz T.A. J. Mol. Biol. 2000; 304: 847-859Crossref PubMed Scopus (191) Google Scholar, 8Parkinson G. Wilson C. Gunasekara A. Ebright Y. Ebright R. Berman H. J. Mol. Biol. 1996; 260: 395-408Crossref PubMed Scopus (233) Google Scholar), whose crystal structure has been determined in the cAMP-bound active state. Comparison of the two structures (Fig. 1) reveals similar folds for both the DNA and effector binding domains but very different domain orientations. In the symmetric structure of active CRP, the DNA binding domains are positioned near to the effector binding domains in such a way as to expose the F helices to properly interact with target DNA. In contrast, the structure of inactive CooA is asymmetric, presumably because of crystal forces (5Lanzilotta 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 (234) Google Scholar). The B chain has an extended structure due to a fusion of the C helix at the dimer interface with the D helix of the DNA binding domain. The A chain has a bend between the C and D helices as in active CRP, but the domain orientation is completely different. In both monomers of inactive CooA, the F helices are buried from solvent and should be inactive for DNA binding. In both CRP and CooA, the C helices are associated in a coiled-coil, and each is in contact with both effector domains. The heme groups of inactive Fe(II) CooA are ligated on one side by His-77 and on the other by the Pro-2 N terminus of the opposite chain. The Pro-2 ligands are displaced when CO binds (9Yamamoto K. Ishikawa H. Takahashi S. Ishimoro K. Morishima I. Nakajima H. Aono S. J. Biol. Chem. 2001; 276: 11473-11476Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). These interconnections between the two chains no doubt account for the cooperativity observed in CO binding (10Coyle C.M. Puranik M. Youn H. Nielsen S.B. Williams R.D. Kerby R.L. Roberts G.P. Spiro T.G. J. Biol. Chem. 2003; 278: 35384-35393Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). The question of how CO binding to the CooA hemes can induce reorientation of the DNA binding domains has been addressed via mutagenesis and spectroscopic studies (10Coyle C.M. Puranik M. Youn H. Nielsen S.B. Williams R.D. Kerby R.L. Roberts G.P. Spiro T.G. J. Biol. Chem. 2003; 278: 35384-35393Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 11Thorsteinsson 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, 12Youn H. Kerby R.L. Clark R.W. Burstyn J.N. Roberts G.P. J. Biol. Chem. 2002; 277: 33616-33623Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 13Thorsteinsson M.V. Kerby R.L. Roberts G.P. Biochemistry. 2000; 39: 8284-8290Crossref PubMed Scopus (23) Google Scholar, 14Youn H. Kerby R.L. Roberts G.P. J. Biol. Chem. 2003; 278: 2333-2340Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 15Yamashita T. Hoashi Y. Tomisugi Y. Ishikawa Y. Uno T. J. Biol. Chem. 2004; 279: 47320-47325Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). From resonance Raman (RR) evidence on altered proteins, we have proposed (10Coyle C.M. Puranik M. Youn H. Nielsen S.B. Williams R.D. Kerby R.L. Roberts G.P. Spiro T.G. J. Biol. Chem. 2003; 278: 35384-35393Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) that the displacement of Pro-2 by CO induces displacement of the hemes into adjacent hydrophobic cavities, accompanied by readjustment of the C helices. The importance of C-helix repositioning has also been shown by a substantial amount of functional analysis of variants altered in this region (16Kerby R.L. Youn H. Thorsteinsson M.V. Roberts G.P. J. Mol. Biol. 2003; 325: 809-823Crossref PubMed Scopus (40) Google Scholar). We now elaborate our model with further evidence including RR studies of variants that are constitutively active (active in the absence of CO) as well as other variants with low activity even in the presence of CO. In addition we suggest a mechanism for the initiation of C-helix bending required for reorientation of the DNA domains through protein tension generated by the heme displacement. Finally we present evidence that CO-bound wild-type (WT) CooA is not entirely in the “on” conformation but is in an intermediate conformation or else in an equilibrating mixture of on and off populations. The latter alternative is supported by previous kinetic measurements (17Puranik M. Nielsen S.B. Youn H. Hvitved A.N. Bourassa J.L. Case M.A. Tengroth C. Balakrishnan G. Thorsteinsson M.V. Groves J.T. McLendon G.L. Roberts G.P. Olson J.S. Spiro T.G. J. Biol. Chem. 2004; 279: 21096-21108Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), indicating that CO-bound CooA is a mixture of forms that are “closed” and “open” with respect to CO dissociation and has recently been discussed more fully in Roberts et al. (3Roberts G.P. Kerby R.L. Youn H. Conrad M. J. Inorg. Biochem. 2005; 99: 280-292Crossref PubMed Scopus (82) Google Scholar). Strains, Plasmids, and in Vivo Assays—The construction of strains overexpressing WT CooA and CooA variants in an Escherichia coli background having a β-galactosidase reporter system in the chromosome was described previously (8Parkinson G. Wilson C. Gunasekara A. Ebright Y. Ebright R. Berman H. J. Mol. Biol. 1996; 260: 395-408Crossref PubMed Scopus (233) Google Scholar). All the site-directed and region-randomized cooA mutations were constructed in a pEXT20-based expression plasmid, which provides tight control of cooA expression (11Thorsteinsson 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). The selection for and properties of 121AYLLRL126 and 121RYLLRL126 CooAs (WT sequence is 121TSCMRT126) are described elsewhere. 5R. L. Kerby, N. D. Lanz, H. Youn, and G. P. Roberts, submitted for publication. Construction and purification of G117I CooA (27Youn H. Kerby R.L. Thorsteinsson M.V. Conrad M. Staples C.R. Serate J. Beack J. Roberts G.P. J. Biol. Chem. 2001; 276: 41603-41610Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) and ΔP3R4 CooA (14Youn H. Kerby R.L. Roberts G.P. J. Biol. Chem. 2003; 278: 2333-2340Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 20Thorsteinsson 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) have been described. CooA Purification—The purification of WT CooA and the variants was performed with our standard method as described previously (12Youn H. Kerby R.L. Clark R.W. Burstyn J.N. Roberts G.P. J. Biol. Chem. 2002; 277: 33616-33623Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar) or by modified protocols (13Thorsteinsson M.V. Kerby R.L. Roberts G.P. Biochemistry. 2000; 39: 8284-8290Crossref PubMed Scopus (23) Google Scholar, 14Youn H. Kerby R.L. Roberts G.P. J. Biol. Chem. 2003; 278: 2333-2340Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). 5R. L. Kerby, N. D. Lanz, H. Youn, and G. P. Roberts, submitted for publication. The purity of WT CooA and CooA variants was estimated to be >95% based on SDS-PAGE. The heme content of CooA preparations was estimated using the extinction coefficient of WT Fe(II)-CO CooA (220 mm-1 cm-1 (31Smulevich G. Mauro J.M. Fishel L.A. English A.M. Kraut J. Spiro T.G. Biochemistry. 1988; 27: 5477-5485Crossref PubMed Scopus (168) Google Scholar)), and protein concentration was measured using the BCA assay (Pierce). In vitro DNA binding assays of WT CooA and CooA variants were performed using the conditions described elsewhere. 5R. L. Kerby, N. D. Lanz, H. Youn, and G. P. Roberts, submitted for publication. As a fluorescence probe, a 26-base pair target DNA containing PcooF was labeled with Texas Red on one end of the duplex and used at the concentration of 6.4 nm. Salmon sperm DNA was used at a 1000-fold excess as the nonspecific DNA competitor. Dissociation constants (Kd) were calculated by fitting of the binding data to a nonlinear equation with correction of the fluorescence quenching as described elsewhere (15Yamashita T. Hoashi Y. Tomisugi Y. Ishikawa Y. Uno T. J. Biol. Chem. 2004; 279: 47320-47325Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Sample Preparation—Purified CooA was diluted in buffer (25 mm MOPS/0.1 m NaCl, pH 7.4) to a heme concentration of ∼20 μm. For AYLLRL and RYLLRL variants, whose solubility and degree of Pro-2 ligation is salt-dependent, 5R. L. Kerby, N. D. Lanz, H. Youn, and G. P. Roberts, submitted for publication. a 25 mm MOPS, 0.5 m NaCl, 50 mm CaCl2, pH 7.4, buffer was used. CooA samples were purged with N2 for 20 min then with CO for ∼5 min followed by reduction of the Fe(III) to the Fe(II) form with sodium dithionite (final concentration, ∼20-60 mm). Reduction and CO binding were monitored by changes in the absorption spectra. The Fe(II) CooA samples were prepared by first purging with N2 for 20 min followed by the reduction with sodium dithionite. To increase the 5-coordinate (5-c) fraction, a buffer with higher salt concentration (25 mm MOPS, 0.5 m NaCl, 50 mm CaCl2, pH 7.4) was used. DNA-bound Fe(II)-CO CooA was prepared according to the published procedures (19Uchida T. Ishikawa H. Takahashi S. Ishimori K. Morishima I. Ohkubo K. Nakajima H. Aono S. J. Biol. Chem. 1998; 273: 19988-19992Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 20Thorsteinsson 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) with the following modifications. The double-stranded DNA sample (provided by Prof. Thomas Poulos, University of California, Irvine), 5′-GCATAACTGTCATCTGGCCGACAGACGATGG-3′ and 3′-GTATTGACAGTAGACCGGCTGTCTGCTACCG-5′, was heated at 80 °C for 5 min in a water bath and then cooled at room temperature for ∼2 h. Then the annealed DNA (final concentration 15 μm) was mixed with a sample of either WT or RYLLRL CooA (final concentration 15 μm) in 40 mm Tris, 50 mm KCl, 6 mm CaCl2, pH 8.0, buffer and incubated at room temperature for ∼30 min. The Fe(II)-CO sample was then prepared as described above. RR Spectroscopy—RR spectra were obtained with excitation wavelengths of 406.7 and 568.1 nm from a Kr+ laser (Spectra Physics, 2080-RS) and 441.6 nm from a He-Cd (Liconix) laser in a 270° backscattering sample geometry. Photodissociation of the bound CO was minimized by using low laser power (∼1 milliwatt at the sample) and by spinning the sample. The scattered light was collected and focused onto a triple spectrograph (Chromex) equipped with a CCD detector (Roper Scientific) operating at 77 K. Spectra were calibrated with dimethyl formamide, ethyl acetate, and dimethyl sulfoxide-d6. In Vitro DNA Binding Analysis Identifies Variants Affected in the Heme Vicinity with a Range of Activities—To explore the connection between ligand binding and protein conformation change, a set of CooA variants was chosen with substitutions predicted to be near the heme. Three classes of variants were investigated; (a) those with changes at position Phe-74, which forms part of the interior heme pocket on the proximal side (His-77, Fig. 1) and is conserved (Phe or Tyr) in CooA homologues (21Youn H. Kerby R.L. Conrad M. Roberts G.P. J. Bacteriol. 2004; 186: 1320-1329Crossref PubMed Scopus (41) Google Scholar), (b) those with changes at position Leu-120, an invariant residue in CooA homologues (21Youn H. Kerby R.L. Conrad M. Roberts G.P. J. Bacteriol. 2004; 186: 1320-1329Crossref PubMed Scopus (41) Google Scholar), which forms part of the heme pocket on the distal side and simultaneously serves as an “a” position coiled-coil residue in the C-helix, and (c) those in which changes to the C-helix position 121-126 segment (Fig. 1) have significantly stabilized the DNA-binding conformation of the protein. 5R. L. Kerby, N. D. Lanz, H. Youn, and G. P. Roberts, submitted for publication. These variants were produced by the mutagenesis scheme described below and selected on the basis of in vivo screens in which CooA activity is linked to the expression of β-galactosidase. 5R. L. Kerby, N. D. Lanz, H. Youn, and G. P. Roberts, submitted for publication. The in vivo activity depends upon the variant accumulation levels, specific DNA binding affinity, and ability to interact with RNA polymerase. Consequently the biochemical properties of the identified variants were examined further through in vitro DNA binding. Randomization of the codon for residue 74 followed by screening for variants with CO-dependent activity in vivo yielded two distinct classes with activity above background (data not shown). Highly active variants possessed either Phe or Tyr residues, whereas variants with lower activity contained Leu, Gly, or His residues. At position 120, similar mutagenesis and screening showed that only Leu afforded CO-dependent activity similar to WT CooA; L120F and L120S, which were chosen for further study, were poorly activated by CO. The selection and analysis of the CO-independent, constitutively active variants possessing changes in the C-helix 121-126 region is elaborated elsewhere. 5R. L. Kerby, N. D. Lanz, H. Youn, and G. P. Roberts, submitted for publication. These changes produce CooA that is active even without added CO. In vitro DNA binding affinity (Table 1) was measured with a fluorescence polarization assay, which monitors binding of CooA to a Texas Red-labeled target DNA (20Thorsteinsson 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). The 121-126 C-helix variants precipitate from solution at high protein concentrations but were stabilized at higher salt concentrations. This property and their exceptional DNA affinity made analysis under standard conditions difficult (50 mm KCl had been used previously (12Youn H. Kerby R.L. Clark R.W. Burstyn J.N. Roberts G.P. J. Biol. Chem. 2002; 277: 33616-33623Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar)); the DNA binding analysis was modified to include higher salt levels (250 mm KCl and 20 mm CaCl2) and a 1000-fold excess of nonspecific DNA to all samples. The affinity of CO-bound WT CooA for the target DNA was decreased ∼10-fold under these conditions relative to assays performed previously at lower salt levels (20Thorsteinsson 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). In terms of DNA binding, F74Y and F74L CooAs showed CO-dependent affinities similar to that of WT CooA (Table 1), suggesting that the poor in vivo activity of F74L CooA represents a defect in its interaction with RNA polymerase. In contrast, changing Leu-120 to Phe or Ser diminished activity in vivo and in vitro, with a >10-fold decrease in DNA affinity relative to CO-bound WT CooA (Table 1). The activities of the 121-126 C-helix variants were unusual in two respects; (a) these variants possessed DNA binding affinities in vitro in the absence of CO that were similar to those of CO-bound WT CooA and (b) in the presence of CO their DNA binding affinity exceeded that of WT CooA. Two rather similar variants in this region were studied, with the following two sequences at positions 121-126: AYLLRL and RYL-LRL. In the following text, we will refer to them by these names or by (A/R)YLLRL for the pair.TABLE 1In vitro DNA binding affinities of purified CooA proteinsCooAFe(II) KdFe(II)-CO Kdnm in hemenm in hemeWTaAverage of independent assays with 95% confidence interval, shown in parentheses. All assays were performed in high salt fluorescence polarization buffer that included 250 mm KCl and 20 mm CaCl2. 5>5,000212 (13)121RYLLRL126aAverage of independent assays with 95% confidence interval, shown in parentheses. All assays were performed in high salt fluorescence polarization buffer that included 250 mm KCl and 20 mm CaCl2. 5178 (18)59 (7)121AYLLRL126 586 (10)46 (2)F74Y>5,000152 (1)F74L>5,000383 (136)L120F>5,000∼2900 (estimated)L120S>5,000∼3200 (estimated)a Average of independent assays with 95% confidence interval, shown in parentheses. All assays were performed in high salt fluorescence polarization buffer that included 250 mm KCl and 20 mm CaCl2. Open table in a new tab RR Frequencies Indicate Variable Fe-His Bond Strengths in CO Adducts of the CooA Variants—Resonance Raman spectra of heme-CO adducts reveal the positions of Fe-C and C-O stretching vibrations, and these positions provide information on both distal and proximal interactions of the adduct with the surrounding protein (22Spiro T.G. Wasbotten I.H. J. Inorg. Biochem. 2005; 99: 34-44Crossref PubMed Scopus (189) Google Scholar). Fig. 2 reveals an unusual pattern for the νFeC and νCO Raman bands of the CooA variants in this study. νCO varies over a significant range, 1975-1986 cm-1, but νFeC is essentially constant, at 486-487 cm-1. When there are changes in back-bonding due electrostatic influences in the vicinity of the bound CO, then νCO and νFeC are anticorrelated; as one increases, the other decreases as a result of changes in back-bonding (22Spiro T.G. Wasbotten I.H. J. Inorg. Biochem. 2005; 99: 34-44Crossref PubMed Scopus (189) Google Scholar). This behavior is illustrated in Fig. 3 (solid line) for a series of myoglobin (Mb) variants having substitutions among residues distal to the bound CO. However, increasing or decreasing the donor strength of the proximal ligand, trans to the CO, produces negative or positive deviations from the back-bonding correlation (22Spiro T.G. Wasbotten I.H. J. Inorg. Biochem. 2005; 99: 34-44Crossref PubMed Scopus (189) Google Scholar). The νCO/νFeC point for WT CooA falls significantly above the Mb line, implying, as noted earlier (10Coyle C.M. Puranik M. Youn H. Nielsen S.B. Williams R.D. Kerby R.L. Roberts G.P. Spiro T.G. J. Biol. Chem. 2003; 278: 35384-35393Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), a significant weakening of the bond to the proximal imidazole ring relative to Mb. This was attributed to displacement of the heme as part of the activation mechanism of CooA induced by CO binding. We now observe that the data for the variants in the present study fall on a horizontal line that intersects the Mb line at about the position occupied by Mb variants in which the distal histidine is replaced by apolar residues. This is exactly the behavior expected if the CO pocket is apolar in CooA (as inferred earlier), but the Fe-His bond is weakened to different extents depending on the variant. The higher νCO reflects diminished back-donation because of diminished forward donation by the His ligand; however, the expected inverse effect on νFeC is compensated by diminished σ competition between the Fe-His and Fe-C bonds (22Spiro T.G. Wasbotten I.H. J. Inorg. Biochem. 2005; 99: 34-44Crossref PubMed Scopus (189) Google Scholar). Density functional theory (DFT) calculations by Franzen (23Franzen S. J. Am. Chem. Soc. 2001; 123: 12578-12589Crossref PubMed Scopus (47) Google Scholar) find νCO lowering but little change in νFeC when the bond to a proximal imidazole is strengthened by donating a H-bond, as does the proximal ligand in Mb (see the lower inset in Fig. 3). We infer that the CooA Fe-His is weakened to various extents, reflecting variable displacement of the heme group. Significantly, νCO is increased when CO-bound WT CooA binds its target DNA sequence, implying a further weakening of the Fe-His bond (Fig. 2). This same higher frequency is seen for CO adducts of the constitutively active (A/R)YLLRL variants, in the absence of DNA, and no further shift is seen when DNA is bound. In the presence of CO, DNA binding affinity is about three times higher for (A/R)YLLRL than for WT CooA (Table 1). We interpret these results to mean that the position 121-126 substitutions have fully stabilized the on conformation of CooA-CO, whereas CO-bound WT CooA is in an equilibrium between on and off forms or else it is in an intermediate form, which is turned on by DNA binding. Although an equilibrium between two forms should produce a superposition of two spectral responses, the spectral resolution is insufficient to distinguish a superposition from an intermediate response; the difference in the central position of the νCO band is less than its width (Fig. 2). An equilibrium mixture of on and off populations would be consistent with kinetic evidence for “open” and “closed” forms of CooA coexisting in the CO-bound WT protein (17Puranik M. Nielsen S.B. Youn H. Hvitved A.N. Bourassa J.L. Case M.A. Tengroth C. Balakrishnan G. Thorsteinsson M.V. Groves J.T. McLendon G.L. Roberts G.P. Olson J.S. Spiro T.G. J. Biol. Chem. 2004; 279: 21096-21108Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). On the other hand the L120S and L120F variants, which display low CO-dependent activity, have significantly stronger Fe-His bonds, as judged by their νCO/νFeC points being closer to the Mb line than is the WT protein. Thus, the low activity of these mutants can be attributed to destabilization of the on conformation, with an attendant diminution in the average heme displacement. The L120S variant displays a small but noticeable (3 cm-1) downshift in νFeC relative to the L120F variant and a corresponding upshift (4 cm-1) in νCO. These opposite shifts are a signal of decreased back-donation, associated with negative polarity among distal groups near the bound CO. This effect implies that the Leu-120 side chain is close to the CO and that replacement with Ser introduces negative polarity via the OH lone pair, similar to the H64V/V68T variant of Mb. Yamashita et al. (15Yamashita T. Hoashi Y. Tomisugi Y. Ishikawa Y. Uno T. J. Biol. Chem. 2004; 279: 47320-47325Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar) reached a similar conclusion by examining the L120N variant, although in that case there was a 6-cm-1 upshift in νFeC, presumably reflecting positive polarity from the asparagine NH2 group (15Yamashita T. Hoashi Y. Tomisugi Y. Ishikawa Y. Uno T. J. Biol. Chem. 2004; 279: 47320-47325Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Aono and co-workers (19Uchida T. Ishikawa H. Takahashi S. Ishimori K. Morishima I. Ohkubo K. Nakajima H. Aono S. J. Biol. Chem. 1998; 273: 19988-19992Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) reported that adding DNA to CooA induced an additional νFeC RR band at 519 cm-1 and a narrowing of the main νFeC band at 487 cm-1.Wedonot observe these effects (Fig. 2) and note that their experiments were carried out without added divalent cations, which have subsequently been shown to be essential for DNA binding (24Youn H. Thorsteinsson M.V. Kerby R.L. Conrad M. Roberts G.P. J. Bacteriol. 2005; 187: 2573-2581Crossref PubMed Scopus (8) Google Scholar). Also, the CooA preparation studied by Aono and co-workers (19Uchida T. Ishikawa H. Takahashi S. Ishimori K. Morishima I. Ohkubo K. Nakajima H. Aono S. J. Biol. Chem. 1998; 273: 19988-19992Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) exhibited the νCO band at 1969 cm-1, much lower than the 1982 cm-1 we observe but close to a subsidiary νCO band seen in our spectra at higher amplification and assigned earlier to a fraction of inactive molecules with undisplaced heme (10Coyle C.M. Puranik M. Youn H. Nielsen S.B. Williams R.D. Kerby R.L. Roberts G.P. Spiro T.G. J. Biol. Chem. 2003; 278: 35384-35393Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). The preparation of Aono and co-workers did contain a fraction with νCO = 1979 cm-1, the position found in picosecond Fourier transform IR experiments for CO-bound CooA molecules undergoing photolysis (25Rubtsov I.V. Zhang T.Q. Nakajima H. Aono S. Rubtsov G.I. Kumazaki S. Yoshihara K. J. Am. Chem. Soc. 2001; 123: 10056-10062Crossref PubMed Scopus (28) Google Scholar). This can be taken to represent the active fraction of molecules. Constitutively Active Variants without CO Show Weakened Fe-His Bonds—To assess the Fe-His bond strength in the absence of bound CO, we determined the frequen" @default.
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