Molecular Basis of the Bohr Effect in Arthropod Hemocyanin
2008; Elsevier BV; Volume: 283; Issue: 46 Linguagem: Inglês
10.1074/jbc.m803433200
ISSN1083-351X
AutoresShun Hirota, Takumi Kawahara, Mariano Beltramini, Paolo Di Muro, Richard S. Magliozzo, J. Peisach, Linda S. Powers, Naoki Tanaka, Satoshi Nagao, Luigi Bubacco,
Tópico(s)Insect Utilization and Effects
ResumoFlash photolysis and K-edge x-ray absorption spectroscopy (XAS) were used to investigate the functional and structural effects of pH on the oxygen affinity of three homologous arthropod hemocyanins (Hcs). Flash photolysis measurements showed that the well-characterized pH dependence of oxygen affinity (Bohr effect) is attributable to changes in the oxygen binding rate constant, kon, rather than changes in koff. In parallel, coordination geometry of copper in Hc was evaluated as a function of pH by XAS. It was found that the geometry of copper in the oxygenated protein is unchanged at all pH values investigated, while significant changes were observed for the deoxygenated protein as a function of pH. The interpretation of these changes was based on previously described correlations between spectral lineshape and coordination geometry obtained for model compounds of known structure (Blackburn, N. J., Strange, R. W., Reedijk, J., Volbeda, A., Farooq, A., Karlin, K. D., and Zubieta, J. (1989) Inorg. Chem., 28, 1349-1357). A pH-dependent change in the geometry of cuprous copper in the active site of deoxyHc, from pseudotetrahedral toward trigonal was assigned from the observed intensity dependence of the 1s → 4pz transition in x-ray absorption near edge structure (XANES) spectra. The structural alteration correlated well with increase in oxygen affinity at alkaline pH determined in flash photolysis experiments. These results suggest that the oxygen binding rate in deoxyHc depends on the coordination geometry of Cu(I) and suggest a structural origin for the Bohr effect in arthropod Hcs. Flash photolysis and K-edge x-ray absorption spectroscopy (XAS) were used to investigate the functional and structural effects of pH on the oxygen affinity of three homologous arthropod hemocyanins (Hcs). Flash photolysis measurements showed that the well-characterized pH dependence of oxygen affinity (Bohr effect) is attributable to changes in the oxygen binding rate constant, kon, rather than changes in koff. In parallel, coordination geometry of copper in Hc was evaluated as a function of pH by XAS. It was found that the geometry of copper in the oxygenated protein is unchanged at all pH values investigated, while significant changes were observed for the deoxygenated protein as a function of pH. The interpretation of these changes was based on previously described correlations between spectral lineshape and coordination geometry obtained for model compounds of known structure (Blackburn, N. J., Strange, R. W., Reedijk, J., Volbeda, A., Farooq, A., Karlin, K. D., and Zubieta, J. (1989) Inorg. Chem., 28, 1349-1357). A pH-dependent change in the geometry of cuprous copper in the active site of deoxyHc, from pseudotetrahedral toward trigonal was assigned from the observed intensity dependence of the 1s → 4pz transition in x-ray absorption near edge structure (XANES) spectra. The structural alteration correlated well with increase in oxygen affinity at alkaline pH determined in flash photolysis experiments. These results suggest that the oxygen binding rate in deoxyHc depends on the coordination geometry of Cu(I) and suggest a structural origin for the Bohr effect in arthropod Hcs. Hemocyanins (Hcs) 3The abbreviations used are: Hc, hemocyanin; XANES, x-ray absorption near edge spectroscopy; XAS, x-ray absorption spectroscopy; CT, charge transfer; SOD, superoxide dismutase; MES, 4-morpholine-ethanesulfonic acid. 3The abbreviations used are: Hc, hemocyanin; XANES, x-ray absorption near edge spectroscopy; XAS, x-ray absorption spectroscopy; CT, charge transfer; SOD, superoxide dismutase; MES, 4-morpholine-ethanesulfonic acid. are oxygen carrier and storage proteins found in molluscs and arthropods. Significant differences are observed between mollusc and arthropod Hcs in size of the functional units and in their tertiary and quaternary structures. Specifically, arthropod Hcs are structurally homogenous oligomeric proteins with a minimal functional subunit of 75 kDa. Under physiological conditions in the presence of calcium, the subunits are typically arranged in hexamers and dodecamers. Removal of Ca2+ with EDTA at neutral pH causes dissociation of the dodecamer into hexamers, which can be dissociated into monomers at alkaline pH (1Dainese E. Di Muro P. Beltramini M. Salvato B. Decker H. Eur. J. Biochem. 1998; 256: 350-358Crossref PubMed Scopus (45) Google Scholar). The different aggregation states are related to modified oxygen binding properties (2Markl J. Decker H. Adv. Comp. Environ. Physiol. 1992; 13: 325-376Crossref Google Scholar). As generally observed for respiratory proteins, a fundamental physiological property of Hc is its competence to bind oxygen with different affinity in response to allosteric effectors including hydrogen ions. Detailed structural information concerning the active site of Hcs is derived mainly from a limited set of x-ray crystallographic investigations. The active site structures of the deoxy form have been described for two arthropod Hcs, Panulirus interruptus (3Volbeda A. Hol W.G. J. Mol. Biol. 1989; 209: 249-279Crossref PubMed Scopus (372) Google Scholar) and Limulus polyphemus (subunit II) (4Hazes B. Magnus C. Bonaventura C. Bonaventura J. Dauter Z. Kalk K.H. Hol W.G. Protein Science. 1993; 2: 597-619Crossref PubMed Scopus (315) Google Scholar). However, a comparison of the results is limited by the different solution conditions from which the proteins were crystallized. In both examples, each Cu(I) of the binuclear active site is coordinated by the ϵ-nitrogen of three histidine imidazole residues. In the P. interruptus structure obtained from protein crystallized at pH 4.8, two of the three histidine imidazole ligands and one copper atom lie nearly in a plane, with Cu-N bond lengths of 1.9 Å. In addition, each metal atom binds to a third histidyl imidazole nitrogen at a distance of 2.7 Å, perpendicular to the plane defined by the two equatorial histidine nitrogens and a copper atom. In contrast to this, the structure of L. polyphemus subunit II deoxyHc, obtained from crystals grown in 0.5 m chloride at pH 6.5-7.0, shows that each copper has approximately trigonal planar coordination geometry, with three histidyl imidazole nitrogens at a distance of 1.9-2.2 Å from the copper. These ligands define a plane from which the copper is displaced by 0.3 Å. An important additional difference between these two structures is the metal-metal distance, which is 3.6 Å in P. interruptus Hc (low pH and low ionic strength) and 4.6 Å for L. polyphemus Hc (physiological pH and higher ionic strength, Fig. 1). The only example of an oxy structure for an arthropod Hc is that of subunit II of the L. polyphemus protein crystallized at pH 6.2 (5Magnus K.A. Hazes E.E. Lattman A. Volbeda A. Hol W.G. Proteins: Struct. Funct. Gen. 1991; 9: 240-247Crossref PubMed Scopus (21) Google Scholar). In this example, the oxygen molecule is bound as a peroxide and forms a μ:η2-η2 bridge between the two Cu(II) atoms. Each copper atom is pentacoordinated in a square pyramidal geometry, where the equatorial plane is now defined by two histidyl imidazole nitrogens and the bound oxygen, while a third histidyl nitrogen is axially coordinated to copper. All three Cu-N bond lengths are 1.9 Å, and the metal-metal separation is 3.0-3.5 Å (6Magnus K.A. Hazes B. Ton-That H. Bonaventura C. Bonaventura J. Hol W.G. Proteins. 1994; 19: 302-309Crossref PubMed Scopus (382) Google Scholar), a distance supported by EXAFS analysis for the closely related P. interruptus oxyHc (7Feiters M.C. Comments Inorg. Chem. 1990; 11: 131-174Crossref Google Scholar, 8Volbeda A. Feiters M.C. Vincent M.G. Bouwman E. Dobson B. Kalk K.H. Reedijk J. Hol W.G. Eur. J. Biochem. 1989; 181: 669-673Crossref PubMed Scopus (13) Google Scholar). Oxygen binding to arthropods Hcs is very sensitive to pH, and the relevance of this Bohr effect resides in the capability of these proteins to respond to a pH variation in the medium with a variation in affinity for molecular oxygen to meet physiological needs. Although Hc has been studied extensively, detailed information concerning oxygen binding kinetics and the origins of both cooperativity and the Bohr effect are still limited (4Hazes B. Magnus C. Bonaventura C. Bonaventura J. Dauter Z. Kalk K.H. Hol W.G. Protein Science. 1993; 2: 597-619Crossref PubMed Scopus (315) Google Scholar, 6Magnus K.A. Hazes B. Ton-That H. Bonaventura C. Bonaventura J. Hol W.G. Proteins. 1994; 19: 302-309Crossref PubMed Scopus (382) Google Scholar). A useful approach for the study of oxygen binding kinetics is flash photolysis. OxyHc is blue and its optical spectrum is characterized by CT bands about 345 nm and at about 600 nm (9Jolley Jr., R.L. Evans L.H. Makino N. Mason H.S. J. Biol. Chem. 1974; 249: 335-345Abstract Full Text PDF PubMed Google Scholar, 10Heirwegh K. Borginon H. Lontie R. Biochim. Biophys. Acta. 1961; 48: 517-526Crossref PubMed Scopus (106) Google Scholar, 11van Holde K.E. Biochemistry. 1967; 6: 93-99Crossref PubMed Scopus (63) Google Scholar, 12Solomon E.I. Lowery M.D. Science. 1993; 259: 1575-1581Crossref PubMed Scopus (400) Google Scholar). DeoxyHc is colorless. Taking advantage of these optical differences, a photolysis approach was previously used to study the binding of oxygen to Streptomyces antibioticus tyrosinase, a monomeric enzyme that like Hc contains a binuclear copper center with similar optical properties (13Hirota S. Kawahara T. Lonardi E. de Waal E. Funasaki N. Canters G.W. J. Am. Chem. Soc. 2005; 127: 17966-17967Crossref PubMed Scopus (15) Google Scholar). In the present study, flash photolysis was used to investigate oxygen binding to Hc allowing us to probe allosteric effects. A complementary approach to the kinetic studies is x-ray absorption spectroscopy (XAS), which allows for the investigation of the immediate coordination geometry of Cu(I) in deoxyHc. For Cu(I), a species with limited potential for other spectroscopic studies as it lacks color and paramagnetism, coordination geometry can be deduced from copper k-edge absorbance measurements. Previous studies on Cu(I) model complexes of known structure provide the background information used to define a correlation between k-edge features and coordination geometry (14Cramer S.P. Eccles T.K. Kutzler F.W. Hodgson K.O. Mortenson L.E. J. Am. Chem. Soc. 1976; 98: 1287-1288Crossref PubMed Scopus (113) Google Scholar, 15Smith T.A. Penner-Hahn J.E. Berding M.A. Doniach S. Hodgson K.O. J. Am. Chem. Soc. 1985; 107: 5945-5955Crossref Scopus (163) Google Scholar). Three paradigmatic arthropod Hcs that are structurally and functionally characterized were used here (2Markl J. Decker H. Adv. Comp. Environ. Physiol. 1992; 13: 325-376Crossref Google Scholar). Flash photolysis was used to obtain the kinetic parameters of oxygen binding, while XAS was used to define structural features of copper centers in the active site as a function of pH. The results suggest that the O2 binding rate depends on the coordination geometry of Cu(I) in deoxyHc providing evidence for a structural origin of the Bohr effect. Sample Preparation—P. interruptus and Carcinus aestuarii Hcs were purified and stored as previously described (1Dainese E. Di Muro P. Beltramini M. Salvato B. Decker H. Eur. J. Biochem. 1998; 256: 350-358Crossref PubMed Scopus (45) Google Scholar, 16Magliozzo R.S. Bubacco L. McCracken J. Jiang F. Beltramini M. Salvato B. Peisach J. Biochemistry. 1995; 34: 1513-1523Crossref PubMed Scopus (15) Google Scholar). L. polyphemus hemolymph was collected at the Marine Biological Laboratories, Woods Hole, MA. The clotted hemolymph was filtered through gauze and centrifuged to eliminate debris. The clear dark blue supernatant was dialyzed overnight at 4 °C against 20 mm CaCl2, pH 7.0. During dialysis, a small amount of precipitate was formed and was separated by low speed centrifugation. The individual subunits were purified according to a published method (17Brenowitz M. Bonaventura C. Bonaventura J. Gianazza E. Arch. Biochem. Biophys. 1981; 210: 748-761Crossref PubMed Scopus (41) Google Scholar). Subunit II was used in this study because of its well-characterized allosteric properties (18Brenowitz M. Bonaventura C. Bonaventura J. Arch. Biochem. Biophys. 1984; 230: 238-249Crossref PubMed Scopus (28) Google Scholar). The purity of the protein was verified by the electrophoretic mobility, while its aggregation state in the presence of Ca2+ was determined by analytical ultracentrifugation using Schlieren optics (18Brenowitz M. Bonaventura C. Bonaventura J. Arch. Biochem. Biophys. 1984; 230: 238-249Crossref PubMed Scopus (28) Google Scholar). Control oxygen binding experiments were carried out using a thin layer optical cell as previously described (19Kraus D.W. Wittemberg J.B. J. Biol. Chem. 1990; 265: 16043-16053Abstract Full Text PDF PubMed Google Scholar). Flash Photolysis Measurements—Purified Hc of C. aestuarii in 50 mm Tris-HCl buffer, pH 8.3, was degassed on a vacuum line and then flushed with nitrogen gas. Hydroxylamine was added to the sample solution anaerobically (final concentration, 1 mm) to reduce any oxidized protein. The solution was then dialyzed under aerobic conditions against 50 mm Tris-HCl, MES, or acetate buffer at the desired pH. After dialysis, the protein concentration was adjusted to 20 μm, and a sample was placed under an atmosphere of oxygen plus nitrogen such that the partial pressure of oxygen could be varied from 5 to 100% with a mixed gas generator (MX-3S, Crown, Tokyo, Japan). The sample solution was filtered under the appropriate oxygen concentration and was transferred into the sealed quartz cell, which was filled with the same gas mixture. Photolysis of oxyHc samples was accomplished using the third harmonic of an Nd:YAG laser (Surelight I-10, Continuum, Santa Clara, pulse energy, 30 mJ; pulse width, 5 ns) for excitation. Time-resolved absorbance changes were measured at 20 °C with illumination from a Xe lamp orthogonal to the laser pulse and were recorded on a digital oscilloscope (TDS 3012B, Tektronix, Tokyo, Japan), which received voltage signals from the photomultiplier attached to a monochromator (RSP-601-03, Unisoku, Osaka, Japan). The traces were obtained as the average of 256 or 512 pulses, and least-squares exponential fits were performed for the time-resolved absorption data using Igor Pro ver. 4.0 (WaveMetrics). X-ray Edge Measurements—Highly concentrated Hc (2 mm copper concentration) used in spectroscopic experiments was prepared using a Centricon filter (Amicon) and then transferred to Lucite® sample holders designed for the x-ray measurements (20Brown J.M. Powers L. Kincaid B. Larrabee J.A. Spiro T.G. J. Am. Chem. Soc. 1980; 102: 4210-4216Crossref Scopus (211) Google Scholar). DeoxyHc was obtained by passing a pre-humidified argon stream through an air tight, homemade chamber designed to host the loaded sample holders. Through the transparent faces (top and bottom) of the chamber, it was possible to monitor the course of deoxygenation based on the disappearance of the blue color of oxyHc. It was estimated that no more than 2-3% oxyHc remained in the apparently colorless sample by visual inspection. After deoxygenation, the sealed chamber was cooled in liquid nitrogen and the frozen sample was removed and stored at 77 K until used. All samples were placed ina25 × 2.5 × 2 mm Lucite® sample holder covered with Kapton® tape. X-ray fluorescence data were collected on beam lines X-9A and X-10C at the National Synchrotron Light Source (NSLS), Brookhaven National Laboratory, using double flat Si(111) (X-9A) and Si(220) (X-10C) crystal monochromators with fixed exit geometry. Beam harmonics were rejected using a nickel (X-9A) or rhodium- (X-10C) coated mirror positioned downstream of the monochromator. The sample temperature was maintained at ∼120 K by flowing cooled nitrogen gas through a Lucite® cryostat as described previously (1Dainese E. Di Muro P. Beltramini M. Salvato B. Decker H. Eur. J. Biochem. 1998; 256: 350-358Crossref PubMed Scopus (45) Google Scholar). X-ray edge data having 3 eV resolution (X-9A) and 2 eV resolution (X-10C) were recorded by counting at single energy values for 3 s and incrementing the energy in 0.5 eV steps from 30 eV below the copper edge to 120 eV above the edge. Copper foil was used as an energy standard to account for any shifts in the monochromator calibration for any individual scan. Kapton® tape was mounted at a 45° angle to the incident x-ray beam to scatter x-ray photons through the copper foil. The photons were then counted with a photomultiplier tube positioned perpendicular to the x-ray beam as previously described (1Dainese E. Di Muro P. Beltramini M. Salvato B. Decker H. Eur. J. Biochem. 1998; 256: 350-358Crossref PubMed Scopus (45) Google Scholar). X-ray flux was 1.0 × 1010 (X-9A) and 1.3 × 1010 photons sec-1 mm-2 at 100 mA beam current (X-10C). X-ray edge data were generally taken in the range of 100-225 mA. K-copper fluorescence was detected with a 13-element solid-state energy-resolving germanium detector, and incident photon scattering was rejected by a nickel filter. Reference signals (incident beam intensity, Io) were collected using a standard ion chamber. To monitor the condition of samples after x-ray exposure, optical spectra in the UV and visible regions were collected after sample dilution in buffer. Sample integrity was verified by the ratio between the absorbance at 280 nm and the charge transfer transition of oxyHc at 345 nm. No significant difference was found in this ratio collected before and after the XAS experiments. X-ray edge spectra were normalized by fitting a function to pre- and post-edge regions of the spectrum and normalizing the edge jump to 1.0 at 9060 eV. To rule out systematic errors, partial sums of the total number of scans were independently fit without any significant differences noted. Flash Photolysis of Oxygen from Oxyhemocyanin—The absorbance change after flash photolysis of C. aestuarii oxyHc was monitored at 337 nm, the absorption maximum of the oxyHc spectrum (Fig. 2). To prove that the observed optical change corresponded to oxygen photolysis and re-binding, the wavelength dependence of the initial absorbance change at several wavelengths within the absorption envelope of oxyHc was measured, and this action spectrum is depicted in the inset of Fig. 2. In the same figure, the difference absorption spectrum between oxyHc and deoxyHc is shown (Fig. 2, inset, dotted line), which was well correlated with the wavelength dependence of the initial absorption change after photolysis. The experimental conditions were designed such that 100% of the Hc was in the oxygenated form before photolysis at all oxygen concentrations tested (1Dainese E. Di Muro P. Beltramini M. Salvato B. Decker H. Eur. J. Biochem. 1998; 256: 350-358Crossref PubMed Scopus (45) Google Scholar). The fraction of deoxyHc present at the beginning of detectable oxygen re-binding was about 5% calculated on the basis of the absorbance change. The time scale of absorbance recovery was in the range of microseconds to submilliseconds. The initial absorbance decrease corresponded to dissociation of oxygen from oxyHc, whereas the complete intensity recovery indicated re-binding of oxygen with no detectable photodamage. The observed absorbance changes at 337 nm were fit successfully with single exponentials for all oxygen concentrations at pH 8.3. Although at this pH in the presence of Ca2+, C. aestuarii Hc is known to be a mixture of 90% dimer of hexamers and 10% hexamers (1Dainese E. Di Muro P. Beltramini M. Salvato B. Decker H. Eur. J. Biochem. 1998; 256: 350-358Crossref PubMed Scopus (45) Google Scholar), there was no observable heterogeneity in the kinetic data. There was likewise no evidence for bi-phasic character that might be expected if the Hc were switching between the R- and T-state upon photolysis. The observed oxygen re-binding rate constant (kobs) increased upon increasing the pH from 6.5 to 8.7 (Fig. 3A). The kobs values at pH 8.3 and 6.5 are plotted in Fig. 3B as a function of oxygen concentration; the dependence of kobs on oxygen concentration was steeper for the measurements at pH 8.3 than that at pH 6.5. As will be discussed later, the ordinate intercept of the plots gave coincident koff values, demonstrating that only the kon values are pH-dependent. The shape of the optical spectrum of oxyHc is pH-independent, such that optical spectra at different pH values are superimposable. Therefore, our flash photolysis approach does not require any correction for pH effects on the spectrum of oxyHc. 4The kobs value also increased on the low pH side of the minimum at 6.5 (Fig. 3A) as has been noted before for an analogous arthropod Hc (23Molon A. Di Muro P. Bubacco L. Vasilyev V. Salvato, Beltramini M. Conze W. Hellmann N. Decker H. Eur. J. Biochem. 2000; 267: 7046-7057Crossref PubMed Scopus (16) Google Scholar). This phenomenon is not analyzed further. The absorbance change upon photolysis of oxyHc at pH 7.1 under O2 concentrations lower than 0.35 mm could not be successfully fit with a single exponential curve (supplemental Fig. S1, depicts the case for [O2] = 0.14 mm), although single exponential fits were adequate for Hc at pH 8.3 under all oxygen concentrations studied, and at pH 7.1 under higher oxygen concentrations ([O2] > 0.49 mm). For the samples at pH 7.1 under oxygen concentrations less than 0.35 mm, double exponential curves were required to fit the data, suggesting a second process is operative under these conditions. The cooperative homohexamers of CaeSS3 subunits of Hc (1Dainese E. Di Muro P. Beltramini M. Salvato B. Decker H. Eur. J. Biochem. 1998; 256: 350-358Crossref PubMed Scopus (45) Google Scholar), exhibited only a single phase at pH 7.5 and [O2] = 1.39 mm, with smaller kobs values than the heterohexameric native protein (CaeSS3 homohexamers, 61 ± 5 ms-1; the heterohexameric native protein 72.2 ± 3.2 ms-1). To calculate thermodynamic parameters for the oxygen re-binding reaction, we studied the temperature dependence of kobs. In the tyrosinase case (13Hirota S. Kawahara T. Lonardi E. de Waal E. Funasaki N. Canters G.W. J. Am. Chem. Soc. 2005; 127: 17966-17967Crossref PubMed Scopus (15) Google Scholar), the initial intensity change in absorbance in the photolysis experiments depended on temperature such that as the temperature increased, the intensity change increased, whereas for Hc, there was no observed temperature dependence of the optical features. These observations suggest that the structure of Hc at the active site is more rigid than that of tyrosinase (13Hirota S. Kawahara T. Lonardi E. de Waal E. Funasaki N. Canters G.W. J. Am. Chem. Soc. 2005; 127: 17966-17967Crossref PubMed Scopus (15) Google Scholar) and that the increase in the oxygen-binding rate constant in Hc as a function of temperature is not due to protein structural changes. A linear Eyring plot was obtained based on the temperature dependence of kon within the range 5 to 25 °C (Fig. 4). The activation enthalpy and entropy of oxygen binding to Hc calculated from the temperature dependence were as follows; ΔH‡ = 7.2 kcal/mol and ΔS‡ = -26 cal/(mol·K), respectively, based on the data in Fig. 4. These values compare reasonably well with those reported for oxygen binding to binuclear copper model complexes (21Karlin K.D. Tolman W.B. Kaderli S. Zuberbühler A.D. J. Mol. Catal. A: Chemical. 1997; 117: 215-222Crossref Scopus (26) Google Scholar). Copper K-edge Spectra of Arthropod Hemocyanin—Oxy and deoxyHc have readily distinguishable x-ray absorption spectra. For example, a comparison of the copper k-edge spectra of oxy and deoxy forms of P. interruptus Hc (Fig. 5), a very close homolog of C. aestuarii Hc, indicates a shift of 2-3 eV to higher energy expected from the increase in the 1s-electron binding energy for the metal ion in the oxy protein because of its cupric character (14Cramer S.P. Eccles T.K. Kutzler F.W. Hodgson K.O. Mortenson L.E. J. Am. Chem. Soc. 1976; 98: 1287-1288Crossref PubMed Scopus (113) Google Scholar). The difference spectrum between deoxy and oxyHc allows for an accurate determination of the peak positions in the edge spectra of the deoxy protein. The two largest absorbance difference peaks, at 8982.7 ± 0.5 and 8985.6 ± 0.5 eV, have been assigned to the 1s → 4pz and 1s → (s+p)* transitions, respectively (15Smith T.A. Penner-Hahn J.E. Berding M.A. Doniach S. Hodgson K.O. J. Am. Chem. Soc. 1985; 107: 5945-5955Crossref Scopus (163) Google Scholar). In a separate study, the intensities of these transitions were correlated with the coordination geometry of Cu(I) based on a systematic analysis of x-ray edge spectra for a collection of model complexes having different coordination geometries but the same tridentate nitrogen ligands (20Brown J.M. Powers L. Kincaid B. Larrabee J.A. Spiro T.G. J. Am. Chem. Soc. 1980; 102: 4210-4216Crossref Scopus (211) Google Scholar, 22Blackburn N.J. Strange R.W. Reedijk J. Volbeda A. Farooq A. Karlin K.D. Zubieta J. Inorg. Chem. 1989; 28: 1349-1357Crossref Scopus (70) Google Scholar, 24Metz M. Solomon E.I. J. Am. Chem. Soc. 2001; 123: 4938-4950Crossref PubMed Scopus (131) Google Scholar). More precisely, the intensity of the 8982.7 eV feature, normalized to the edge jump, was found to be inversely proportional to the mean displacement along the z-axis of Cu(I) from the plane defined by the three directly coordinated nitrogen atoms. The intensity is significantly higher for the metal ion coordinated in a trigonal planar geometry compared with that for pseudotetrahedral geometry, in which the copper atom lies out of the plane defined by the ligands (20Brown J.M. Powers L. Kincaid B. Larrabee J.A. Spiro T.G. J. Am. Chem. Soc. 1980; 102: 4210-4216Crossref Scopus (211) Google Scholar, 24Metz M. Solomon E.I. J. Am. Chem. Soc. 2001; 123: 4938-4950Crossref PubMed Scopus (131) Google Scholar). Therefore, the 8982.7 eV feature can serve as a spectroscopic probe of coordination geometry in Cu(I) sites with all nitrogen coordination as in deoxyHc and was used here to define the structural modifications induced by changes in pH. The k-edge spectra for P. interruptus deoxyHc at different pH values are collected in Fig. 6. The oxygen affinity of this Hc increases ∼35-fold from pH 5 to 9 (Table 1) (25Kuiper H.A. Coletta M. Zolla L. Chiancone E. Brunori M. Biochim. Biophys. Acta. 1980; 626: 412-416Crossref PubMed Scopus (16) Google Scholar, 26Kuiper H.A. Gastra W. Beintema J.J. van Bruggen E.F. Schepman A.M. Drenth J. J. Mol. Biol. 1975; 99: 619-629Crossref PubMed Scopus (82) Google Scholar). At pH 5.25, the intensity of the 8982.7 eV feature is low (Fig. 6a). As the pH is raised to 7, and finally to 9, the intensity increases (Fig. 6, b and c). This spectroscopic change is indicative of the alteration of Cu(I) geometry from pseudotetrahedral toward trigonal (22Blackburn N.J. Strange R.W. Reedijk J. Volbeda A. Farooq A. Karlin K.D. Zubieta J. Inorg. Chem. 1989; 28: 1349-1357Crossref Scopus (70) Google Scholar). The spectral changes observed are not due to oxidation of Cu(I) to Cu(II) because the lineshape of the x-ray absorption edge spectra collected at different pH values overlay in the 9000 eV region, with no indication of an energy shift expected if Cu(I) were oxidized to Cu(II). A smaller increase in the intensity of the 8982.7 eV feature with increasing pH was noted for subunit II of L. polyphemus Hc (Fig. 7) compared with the data described for P. interruptus Hc. This protein also has a Bohr effect (18Brenowitz M. Bonaventura C. Bonaventura J. Arch. Biochem. Biophys. 1984; 230: 238-249Crossref PubMed Scopus (28) Google Scholar), and the structural modulation induced by pH is considered here to be analogous to that described for P. interruptus Hc, indicating the general nature of the observations and also establishing a correlation between the magnitude of the Bohr effect and the observed intensity changes for two different Hcs. C. aestuarii, P. interruptus, and L. polyphemus Hcs are highly homologous and show the same trend in the pH dependence of oxygen binding.TABLE 1Values of P50 for oxygen binding to Hcs as a function of pH in the presence of 10 mm CaCl2pHP50 O2 mmHgP. interruptus5.25172aIn 100 mm Bis-Tris, pH 5.25 (30).742bIn 50 mm Tris-HCl, pH 7 and in 50 mm ethanolamine, pH 9 (31).95bIn 50 mm Tris-HCl, pH 7 and in 50 mm ethanolamine, pH 9 (31).C. aestuarii7.144.3cIn 50 mm Tris-HCl (1).7.533.7cIn 50 mm Tris-HCl (1).7.913.6cIn 50 mm Tris-HCl (1).8.36.81cIn 50 mm Tris-HCl (1).Subunit II L. polyphemus72.5dIn 100 mm Tris-HCl, pH 7 and 9 (23).91.4dIn 100 mm Tris-HCl, pH 7 and 9 (23).a In 100 mm Bis-Tris, pH 5.25 (30Brouwer M. Bonaventura C. Bonaventura J. Biochemistry. 1978; 17: 2148-2154Crossref PubMed Scopus (75) Google Scholar).b In 50 mm Tris-HCl, pH 7 and in 50 mm ethanolamine, pH 9 (31Sabatucci A. Ascone I. Bubacco L. Beltramini M. Di Muro P. Salvato B. J. Biol. Inorg. Chem. 2002; 7: 120-128Crossref PubMed Scopus (11) Google Scholar).c In 50 mm Tris-HCl (1Dainese E. Di Muro P. Beltramini M. Salvato B. Decker H. Eur. J. Biochem. 1998; 256: 350-358Crossref PubMed Scopus (45) Google Scholar).d In 100 mm Tris-HCl, pH 7 and 9 (23Molon A. Di Muro P. Bubacco L. Vasilyev V. Salvato, Beltramini M. Conze W. Hellmann N. Decker H. Eur. J. Biochem. 2000; 267: 7046-7057Crossref PubMed Scopus (16) Google Scholar). Open table in a new tab FIGURE 7K-edge x-ray absorption spectra of subunit II of L. polyphemus deoxyHc.a, pH 6 in 10 mm Tris-SO4 buffer; b, pH 9 in 100 mm Tris-SO4 buffer; c, pH 9 in 50 mm Tris-SO4 buffer plus 500 mm NaCl; d, pH 6 in 50 mm Tris-SO4 buffer plus 500 mm NaCl. CaCl2 (20 mm) was present in all samples. The buffer concentrations for a and b were chosen to compensate for ionic strength effects.View Large
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