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• W2025531019 abstract "An interaction between cytochrome a in oxidized cytochrome c oxidase (CcO) and anions has been characterized by EPR spectroscopy. Those anions that affect the EPR g = 3 signal of cytochrome a can be divided into two groups. One group consists of halides (Cl-, Br-, and I-) and induces an upfield shift of the g = 3 signal. Nitrogen-containing anions (CN-, NO2-, N3-, NO3-) are in the second group and shift the g = 3 signal downfield. The shifts in the EPR spectrum of CcO are unrelated to ligand binding to the binuclear center. The binding properties of one representative from each group, azide and chloride, were characterized in detail. The dependence of the shift on chloride concentration is consistent with a single binding site in the isolated oxidized enzyme with a Kd of ∼3 mm. In mitochondria, the apparent Kd was found to be about four times larger than that of the isolated enzyme. The data indicate it is the chloride anion that is bound to CcO, and there is a hydrophilic size-selective access channel to this site from the cytosolic side of the mitochondrial membrane. An observed competition between azide and chloride is interpreted by azide binding to three sites: two that are apparent in the x-ray structure plus the chloride-binding site. It is suggested that either Mg2+ or Arg-438/Arg-439 is the chloride-binding site, and a mechanism for the ligand-induced shift of the g = 3 signal is proposed. An interaction between cytochrome a in oxidized cytochrome c oxidase (CcO) and anions has been characterized by EPR spectroscopy. Those anions that affect the EPR g = 3 signal of cytochrome a can be divided into two groups. One group consists of halides (Cl-, Br-, and I-) and induces an upfield shift of the g = 3 signal. Nitrogen-containing anions (CN-, NO2-, N3-, NO3-) are in the second group and shift the g = 3 signal downfield. The shifts in the EPR spectrum of CcO are unrelated to ligand binding to the binuclear center. The binding properties of one representative from each group, azide and chloride, were characterized in detail. The dependence of the shift on chloride concentration is consistent with a single binding site in the isolated oxidized enzyme with a Kd of ∼3 mm. In mitochondria, the apparent Kd was found to be about four times larger than that of the isolated enzyme. The data indicate it is the chloride anion that is bound to CcO, and there is a hydrophilic size-selective access channel to this site from the cytosolic side of the mitochondrial membrane. An observed competition between azide and chloride is interpreted by azide binding to three sites: two that are apparent in the x-ray structure plus the chloride-binding site. It is suggested that either Mg2+ or Arg-438/Arg-439 is the chloride-binding site, and a mechanism for the ligand-induced shift of the g = 3 signal is proposed. The respiratory heme-copper oxidases constitute a super-family of terminal oxidases in both prokaryotic organisms and the mitochondria of eukaryotic cells. Cytochrome c oxidases (CcO) 1The abbreviations used are: CcO, cytochrome c oxidase; CcO·CN, complex of oxidized CcO with cyanide; ET, electron transfer; DM, n-dodecyl-β-d-maltoside; Mes, 2-(N-morpholino)ethanesulfonic acid; Ches, 2-(cyclohexylamino)ethanesulfonic acid. 1The abbreviations used are: CcO, cytochrome c oxidase; CcO·CN, complex of oxidized CcO with cyanide; ET, electron transfer; DM, n-dodecyl-β-d-maltoside; Mes, 2-(N-morpholino)ethanesulfonic acid; Ches, 2-(cyclohexylamino)ethanesulfonic acid. catalyze the reduction of molecular oxygen to water using the reducing equivalents supplied by ferrocytochrome c. Four redox centers of CcO are involved in promoting electron transfer (ET) from cytochrome c to dioxygen. CuA and cytochrome a are primary electron acceptors, and electrons from these sites are delivered to the binuclear center of CcO, consisting of cytochrome a3 and CuB, where oxygen is reduced to water. ET is coupled to the generation of transmembrane proton gradient. Two different processes contribute to the formation of this gradient. The first is the oxidation of cytochrome c from the cytosolic side with the protons required for water formation taken from the matrix side. The second process involves active proton translocation from the matrix to the cytosolic side and is referred to as proton pumping. Substantial insight into the catalytic mechanism of the enzyme has been obtained from studies of the interaction of ligands with CcO. Most of these studies are devoted to the direct interaction of external molecules with the redox active centers. However, there are sites distant from the redox centers that may well be involved in catalysis. One such site is a surface segment from Gly-49 to Asn-55 on the cytosolic side of subunit I of bovine CcO. It has been established that this region undergoes a redox-coupled conformational change (1Yoshikawa S. Shinzawa-Itoh K. Nakashima R. Yaono R. Yamashita E. Inoue N. Yao M. Fei M.J. Libeu C.P. Mizushima T. Yamaguchi H. Tomizaki T. Tsukihara T. Science. 1998; 280: 1723-1729Crossref PubMed Scopus (956) Google Scholar, 2Muramato K. Aoyama H. Mochizuki M. Shinzawa-Itoh K. Yamashita E. Yao M. Tsukihara T. Yoshikawa S. Biochim. Biophys. Acta. 2002; 12: 112Google Scholar), and a surface site, Asp-51 of subunit I, sensitive to the redox state of cytochrome a, was identified as essential for proton translocation (3Shimokata K. Katayama Y. Shimada H. Yoshikawa S. Biochim. Biophys. Acta. 2002; 12: 105Google Scholar, 4Tsukihara T. Shimokata K. Katayama Y. Shimada H. Muramato K. Aoyama H. Mochizuki M. Shinzawa-Itoh K. Yamashita E. Yao M. Ishimura Y. Yoshikawa S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15304-15309Crossref PubMed Scopus (357) Google Scholar). A second intriguing distant site is the nonredoxactive magnesium located at the bottom of a well connected to the cytosolic side of the membrane. Magnesium has been suggested to participate in the exit of water from CcO (5Tsukihara T. Aoyama H. Yamashita E. Tomizaki T. Yamaguchi H. Shinzawa-Itoh K. Nakashima R. Yaono R. Yoshikawa S. Science. 1996; 272: 1136-1144Crossref PubMed Scopus (1887) Google Scholar). This suggestion is consistent with the present evidence showing that water departs the catalytic center via a discrete pathway involving magnesium (6Florens L. Schmidt B. McCracken J. Ferguson-Miller S. Biochemistry. 2001; 40: 7491-7497Crossref PubMed Scopus (32) Google Scholar, 7Schmidt B. McCracken J. Ferguson-Miller S. Proc. Natl. Acad. Sci. U. S. A. 2003; (in press)Google Scholar). The experimental identification of distant sites that may have a physiologically relevant interaction with the redox center(s) brings a new aspect for both the consideration and exploration of these sites in the catalytic mechanism. At least one site has been implicated by the modulation of the EPR signal of oxidized cytochrome a by several anions (8Hartzell C.R. Beinert H. Biochim. Biophys. Acta. 1974; 368: 318-338Crossref PubMed Scopus (161) Google Scholar, 9Wilson D.F. Erecinska M. Owen C. Arch. Biochem. Biophys. 1976; 175: 160-172Crossref PubMed Scopus (49) Google Scholar, 10Beinert H. Hansen R.E. Hartzell C.R. Biochim. Biophys. Acta. 1976; 423: 339-355Crossref PubMed Scopus (67) Google Scholar, 11Schoonover J.R. Palmer G. Biochemistry. 1991; 30: 7541-7550Crossref PubMed Scopus (37) Google Scholar, 12Moody A.J. Richardson M. Spencer J.P.E. Brandt U. Rich P. Biochem. J. 1994; 302: 821-826Crossref PubMed Scopus (10) Google Scholar). Because the binuclear center, composed of cytochrome a3 plus CuB, is the established site where external ligands are bound directly, it led to the idea that the EPR signal of cytochrome a is affected from this center. However, our previous work on ligand binding suggested the interaction of cytochrome a with azide is not mediated by the cytochrome a3-CuB center and that there has to be at least one additional binding site (13Li W. Palmer G. Biochemistry. 1993; 32: 1833-1834Crossref PubMed Scopus (58) Google Scholar). This site was recently located in the crystal structure of the enzyme-azide complex and shown to be in the proximity of cytochrome a on the hydrophobic surface of the enzyme within the membrane (14Fei M.J. Yamashita E. Inoue N. Yao M. Yamaguchi H. Tsukihara T. Shinzawa-Itoh K. Nakashima R. Yoshikawa S. Acta Cryst. D. 2000; 56: 529-535Crossref PubMed Scopus (34) Google Scholar). In this work we have used EPR spectroscopy to classify anions into three groups via their effect on the low field g-value of cytochrome a in oxidized CcO. One group consists of very weakly or noninteracting anions (phosphate, sulfate). The anions from the other two groups induce opposite shifts of the g = 3 signal. A detailed characterization of the interaction with chloride and azide shows there is a novel chloride-binding site in native oxidized CcO. Hepes, Tris, Mes, Ches, NaN3, l-histidine, HCl, and EDTA were from Sigma. NaCl, Na2SO4, Na2HPO4, and K3Fe(CN)6 were from Fisher. NaCN, KBr, KNO3, and NaNO2 were from Mallinckrodt Chemical Works. KI was from J. T. Baker. Peroxide-free detergent Triton X-100 was from Roche Diagnostics, and n-dodecyl-β-d-maltoside (DM) was from Anatrace. Bovine heart mitochondria and CcO were isolated using the method of Soulimane and Buse (15Soulimane T. Buse G. Eur. J. Biochem. 1995; 227: 588-595Crossref PubMed Scopus (81) Google Scholar) with a minor modification (16Fabian M. Palmer G. FEBS Lett. 1998; 422: 1-4Crossref PubMed Scopus (17) Google Scholar). Briefly, mitochondria were subjected to protein extraction twice with Triton X-100 and K2SO4. The second extract contained solubilized CcO and was further purified on a Sepharose Q fast flow column. In this study, we made some additional changes. EDTA (1 mm) and histidine (1 mm) was added to the original extraction medium (10 mm Tris, pH 7.6, 250 mm sucrose) to minimize binding of transition metals; the EDTA was omitted for the second extraction. After loading on the column the second extract was washed with 1 liter of 10 mm Tris, pH 7.6, 1 mm histidine, and 0.1% Triton X-100 before eluting with a sulfate gradient. (Either K2SO4 or Na2SO4 can be used without any noticeable differences in the binding of anions to isolated CcO.) The preparation of mitochondria depleted of cytochrome c was based on the earlier methods (17Jacobs E.E. Sanadi D.R. J. Biol. Chem. 1960; 235: 531-534Abstract Full Text PDF PubMed Google Scholar, 18Lenaz G. MacLennan D. Methods Enzymol. 1967; 10: 499-504Crossref Scopus (13) Google Scholar). Mitochondria were isolated from fresh beef hearts (15Soulimane T. Buse G. Eur. J. Biochem. 1995; 227: 588-595Crossref PubMed Scopus (81) Google Scholar) and washed twice with 10 mm Tris, pH 7.6, and 250 mm sucrose. The washed mitochondria were diluted to a protein concentration of about 3.3 mg/ml with cold 10 mm Na2SO4, stirred at 4 °C for 30 min, and centrifuged at 26,000 × g for 30 min. Cytochrome c was extracted by suspension of the sediment in cold solution of 100 mm Na2SO4 and 10 mm K3Fe(CN)6. This suspension was stirred at 4 °C for 10 min and centrifuged at 48,000 × g for 25 min. To ensure that extraction of cytochrome c was complete, this step was repeated four times using 100 mm Na2SO4 with ferricyanide omitted and three times with 10 mm Hepes, pH 7.6, containing 50 mm Na2SO4. These mitochondria were used directly for chloride binding measurement at pH 7.6. For experiments at pH 6.8, the pH of the mitochondrial suspension was lowered by small additions of dilute H2SO4. Enzyme concentration was determined at pH 8.0 from the absorbance of isolated oxidized enzyme at 424 nm using A = 158 mm-1 cm-1. Enzyme concentration in the mitochondria was determined on mitochondria clarified with 0.1% dodecyl maltoside from the absorption of dithionite-reduced CcO·CN minus oxidized CcO·CN using ΔA606-620 = 18.6 mm-1 cm-1 for cytochrome a (19Liao G.-L. Palmer G. Biochim. Biophys. Acta. 1996; 1274: 109-111Crossref PubMed Scopus (75) Google Scholar). To determine the dependence of CcO catalytic activity on NaCl concentration, both purified enzyme and mitochondria solubilized with 0.1% DM were used. The oxidation of 10 μm ferrocytochrome c by 4.5 nm CcO was monitored at 550 nm and 22 °C (10 mm Tris, pH 7.6, 0.1% DM). The ionic strength of the solution was held constant (I = 210) using mixtures of Na2SO4 and NaCl. The midpoint potential of cytochrome a was estimated in the CcO·CN under argon atmosphere using cytochrome c present as a redox indicator (Eobs0 = 264 mV at pH 7.7 and 23 °C) (20Margalit R. Schejter A. Eur. J. Biochem. 1973; 32: 492-499Crossref PubMed Scopus (185) Google Scholar). The midpoint potential of cytochrome a was calculated from the equilibrium ratios of (cytochrome a3+)/(cytochrome a2+) and (cytochrome c3+)/(cytochrome c2+). These ratios were obtained from optical spectra after addition of 12.8 μm ferrocytochrome c to 2.6 μm CcO·CN in 10 mm Tris, pH 7.6, and 0.1% Triton X-100. The CcO·CN had been depleted of free cyanide by gel filtration. To determine the effect of chloride on the rate of cytochrome a reduction by ferrocytochrome c at constant and high ionic strength, the CcO·CN complex was used once more. Typically, 3 μm CcO·CN was mixed with 29 μm reduced cytochrome c in the Gibson-Durrum stopped-flow instrument at 13 °C. The buffer was 10 mm Hepes, pH 7.6, and 0.1% DM, and the ionic strength was maintained at I = 460 by NaCl and Na2SO4. The high ionic strength and reduced temperature were necessary to detect the electron transfer kinetics by stopped flow (21Antalis T.M. Palmer G. J. Biol. Chem. 1982; 257: 6194-6206Abstract Full Text PDF PubMed Google Scholar). To determine the effect of chloride on intramolecular electron transfer from cytochrome a to cytochrome a3, 3.7 μm oxidized CcO was rapidly mixed with 40 μm ferrocytochrome c plus 0.1 m dithionite in the stopped-flow instrument at 23 °C under argon. Under these conditions, a large fraction of CuA and cytochrome a was reduced in the dead time of the instrument, and, predominantly, the kinetics of electron transfer to the binuclear center was observed. The solution contained 10 mm Ches buffer, pH 9.0, 0.1% DM, and 48 mm NaCl. In the control samples, chloride was replaced by 16 mm Na2SO4. The stopped-flow measurements were made about 20 min after dissolving stock CcO in the buffer. All samples for EPR were frozen by quick immersion in a methanol dry ice bath at a temperature of -58 °C and then transferred to liquid nitrogen and stored at this temperature. EPR spectra were recorded at 10 K on a Varian E6 spectrometer. The conditions for measurements were: frequency, 9.26 GHz; power, 3 mW; modulation amplitude, 10 G; and the modulation frequency, 100 kHz. All dissociation constants were established from the spectral shift of the g = 3 signal in the EPR spectrum of oxidized CcO induced by the binding of anions. Chloride binding to isolated CcO was studied at three pH values: at pH 5.7 (20 mm Mes, 100 mm Na2SO4, 0.1% Triton X-100), at pH 7. 6 (10 mm Hepes, 0.1% Triton X-100), and at pH 8.8 (10 mm Ches, 0.1% Triton X-100). At pH 5.7, a high ionic strength was necessary to avoid enzyme precipitation. The dependences of spectral shifts on the ligand concentration were normalized before the fit was applied. To normalize the amplitudes, the following assumptions based on the resolutions of the g = 3 peak into two gaussian curves were employed. In mitochondria, the whole population of CcO is in the chloride-bound state at a chloride concentration of 1 m. For purified CcO, about 80% of CcO is in this chloride-bound state in the presence of 20 mm NaCl. In the presence of 20 mm azide, 1 m NaCl converts ∼70% of purified CcO to the chloride-bound state. At liquid helium temperatures, the EPR spectrum of oxidized “rapid” CcO only exhibits signals caused by low spin cytochrome a and CuA. The absorption-like g = 3 signal of oxidized cytochrome a of purified CcO (Fig. 1A) is unaffected by sodium ions over the range 10–400 mm at pH 7.6. This peak is also insensitive to the addition of 100 mm Na2SO4 or 100 mm sucrose, brief sonication of the enzyme under a stream of nitrogen on ice, repeated freezing and thawing, and storage on ice for 14 h in 10 mm Tris, pH 7.6, 100 mm Na2SO4, 0.1% Triton X-100 (w/v). In addition, the complex between cytochrome c and CcO prepared by stoichiometric addition of oxidized cytochrome c to CcO at low ionic strength (10 mm Hepes, pH 7.6, 0.1% Triton X-100) did not affect the peak at g = 3. A downfield shift of about 5 G and slight broadening of the peak occurred following the addition of 150 mm sodium phosphate (Table I).Table IThe effect and affinity of anions to cytochrome a in oxidized CcOLigandsInduced shift of g3 signalKdgaussmmLow-field NaCN302.3 NaN33515.6 NaNO2353.3 NaNO328NDaNot determinedHigh-field NaCl243.5 KBr28∼2bEstimated value KI17∼15bEstimated valueNo or small low-field shift NaPi5 Na2SO40a Not determinedb Estimated value Open table in a new tab However, we have found anions that markedly influence the g = 3 signal of oxidized CcO. On the basis of the observed spectral shifts, all ligands tested were consequently divided into one of three groups (Table I). The first group, nitrogen-containing ligands (cyanide, azide, nitrite, and nitrate), induces a low field shift of the g = 3 peak of ∼30–35 G. The second group contained halides (chloride, bromide, and iodide), which shifted the g = 3 peak by ∼17–30 G to a higher magnetic field. The third group was the large anions, phosphate and sulfate, which caused little, if any, shift. The presence of two opposing spectral effects suggested the presence of two separate binding sites for these ligands. To test this possibility we selected, for further characterization, a representative from each of the first two groups: azide, because it has been located in the vicinity of cytochrome a in the crystal structure of the CcO-azide complex (14Fei M.J. Yamashita E. Inoue N. Yao M. Yamaguchi H. Tsukihara T. Shinzawa-Itoh K. Nakashima R. Yoshikawa S. Acta Cryst. D. 2000; 56: 529-535Crossref PubMed Scopus (34) Google Scholar), and chloride, because it may have some relevance for CcO catalysis (22Orii Y. Mogi T. Sato-Watanabe M. Hirano T. Anraku Y. Biochemistry. 1995; 34: 1127-1132Crossref PubMed Scopus (23) Google Scholar, 23Hirano T. Mogi T. Tsubaki M. Hori H. Orii Y. Anraku Y. J. Biochem. 1997; 122: 430-437Crossref PubMed Scopus (12) Google Scholar). Chloride Binding—It has been demonstrated previously that chloride can bind to the binuclear center of CcO (24Scott R.A. Li P.M. Chan S.I. Ann. N. Y. Acad. Sci. 1988; 550: 53-58Crossref PubMed Scopus (34) Google Scholar, 25Moody A.J. Cooper C.E. Rich P. Biochim. Biophys. Acta. 1991; 1059: 189-207Crossref PubMed Scopus (116) Google Scholar, 26Butler C.S. Seward H.E. Greenwood C. Thomson A.J. Biochemistry. 1997; 36: 16259-16266Crossref PubMed Scopus (69) Google Scholar, 27Giuffre A. Stubauer G. Brunori M. Sarti P. Torres J. Wilson M.T. J. Biol. Chem. 1998; 273: 32475-32478Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 28Ralle M. Verkhovskaya M.L. Morgan J.E. Verkhovsky M.I. Wikström M. Blackburn N.J. Biochemistry. 1999; 38: 7185-7194Crossref PubMed Scopus (52) Google Scholar, 29Fabian M. Skultety L. Brunel C. Palmer G. Biochemistry. 2001; 40: 6061-6069Crossref PubMed Scopus (23) Google Scholar, 30Forte E. Barone M.C. Brunori M. Sarti P. Giuffre A. Biochemistry. 2002; 41: 13046-13052Crossref PubMed Scopus (25) Google Scholar). Thus, the observation that the g = 3 peak of cytochrome a is sensitive to chloride raises the question of whether or not the influence of chloride on the EPR spectrum of cytochrome a is a consequence of chloride binding to the catalytic center. That this is not the case was established by the following observations. First, the rate of chloride binding to the site that interacts with cytochrome a is much faster than the rate of chloride binding to the binuclear center (29Fabian M. Skultety L. Brunel C. Palmer G. Biochemistry. 2001; 40: 6061-6069Crossref PubMed Scopus (23) Google Scholar). The reaction with cytochrome a was complete in <1 min, the time needed for manually mixing the enzyme and chloride and freezing the EPR sample in a methanol/dry ice slush. Longer incubation of CcO with chloride at room temperature had no additional affect on the EPR spectrum. By contrast, the binding of chloride to the binuclear center occurs on a time scale of hours at pH 7.6 (29Fabian M. Skultety L. Brunel C. Palmer G. Biochemistry. 2001; 40: 6061-6069Crossref PubMed Scopus (23) Google Scholar). Second, the g = 3 signal observed in the presence of chloride is restored to the signal of the original untreated CcO immediately following the removal of chloride by gel filtration, whereas release of chloride from the binuclear center is an extremely slow process (29Fabian M. Skultety L. Brunel C. Palmer G. Biochemistry. 2001; 40: 6061-6069Crossref PubMed Scopus (23) Google Scholar). Third, the binding of chloride to the binuclear center is associated with the changes in the optical spectrum (12Moody A.J. Richardson M. Spencer J.P.E. Brandt U. Rich P. Biochem. J. 1994; 302: 821-826Crossref PubMed Scopus (10) Google Scholar, 27Giuffre A. Stubauer G. Brunori M. Sarti P. Torres J. Wilson M.T. J. Biol. Chem. 1998; 273: 32475-32478Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 29Fabian M. Skultety L. Brunel C. Palmer G. Biochemistry. 2001; 40: 6061-6069Crossref PubMed Scopus (23) Google Scholar) that were not observed when the EPR spectrum of cytochrome a was already modified by chloride. Finally, the influence of chloride and all other ligands tested on the cytochrome a EPR signal was also observed in the complex of oxidized CcO with cyanide bound in the binuclear center. Increasing the concentration of sodium chloride in samples of purified oxidized CcO led to an increasing amount of enzyme with its g = 3 peak shifted upfield by about 24 gauss (Fig. 1A). Thus, in a sample containing 20 mm NaCl, the EPR spectrum was resolved into two gaussian curves with about 80% of CcO having the upfield shifted signal (chloride-bound) and the remainder chloride-free. Between 0 and 20 mm NaCl the binding process is associated with a decrease of EPR intensity of unreacted enzyme with a parallel increase of intensity because of chloride-bound enzyme. This process can be nicely visualized from the difference EPR spectra of chloride-treated minus untreated enzyme (Fig. 1B). At concentrations of sodium chloride between 20 mm and 1 m, the effect of chloride was reversed, and the normalized amplitude in the difference spectra slightly decreased (Fig. 2A). A similar dependence was obtained from normalized areas under gaussian curves (Fig. 2A). For this purpose, the g = 3 peak at each chloride concentration was resolved to two gaussian curves and the relative area under the curve, corresponding to the chloride-bound state, calculated. The dependence of normalized amplitudes can be fitted assuming a single chloride-binding site with a Kd of 3.5 mm at pH 7.6 (Fig. 2B). This Kd was insensitive to increasing the ionic strength by the addition of 50 mm Na2SO4, to replacing the detergent Triton X-100 with 0.1% DM, and to changing of the buffer from 10 mm Hepes to 10 mm Tris. The estimated Kd for bromide and iodide are 2 and 15 mm, respectively (Table I). Measurements with chloride at both pH 5.7 and 8.8 gave almost identical results. In both cases, the binding of chloride proceeded in two phases, with the first phase having Kd values of 3.4 and 2.5 mm at pH 5.7 and 8.8, respectively. At all three pH values (pH = 5.7, 7.6, and 8.8), raising the concentration of NaCl to 1 m did not convert the g = 3 peak of isolated CcO to a homogenous EPR signal. At this high concentration of NaCl, ∼70% of the enzyme population exists in the high field-shifted state. Chloride has a similar effect on the EPR signal of oxidized CcO present in mitochondrial membranes (8Hartzell C.R. Beinert H. Biochim. Biophys. Acta. 1974; 368: 318-338Crossref PubMed Scopus (161) Google Scholar). Because the EPR spectra of cytochrome c and cytochrome a overlap in the g = 3 region, we first depleted the mitochondria of cytochrome c as described under “Experimental Procedures.” With membrane-bound CcO, we noticed two differences in the behavior of the g = 3 signal compared with the isolated enzyme. First, there was only the high field shift of the g = 3 signal upon chloride binding; the second phase of chloride binding is missing at both pH 7.6 and 6.8. Second, the dissociation constant is about 4–5-fold larger than that found with the purified enzyme. From the fits to the data for CcO in mitochondria, a single binding site with Kd values of 12.5 and 15.6 mm at pH values of 7.6 (Fig. 2B) and 6.8, respectively, were determined. The presence of a second phase in chloride binding with isolated CcO, but not with the membrane-bound enzyme, suggests the purification procedure leads to some modification of the enzyme. To examine this possibility, we collected samples of enzyme after each purification step and measured the EPR spectra of the untreated sample and the sample treated with 200 mm NaCl. The data suggested that CcO is modified during chromatography on Sepharose Q. Thus, the longer the time the enzyme spends being washed on the Sepharose Q column (with either 70 mm K2SO4 or Na2SO4 in 10 mm Tris, pH 7.6, and 0.1% Triton X-100), the greater the fraction of CcO that reacts with chloride in two phases. Competition between the Binding of Azide and Chloride— The crystal structure of the azide complex of bovine cytochrome oxidase reveals a second binding site for azide that is close to cytochrome a and on the surface of the enzyme (14Fei M.J. Yamashita E. Inoue N. Yao M. Yamaguchi H. Tsukihara T. Shinzawa-Itoh K. Nakashima R. Yoshikawa S. Acta Cryst. D. 2000; 56: 529-535Crossref PubMed Scopus (34) Google Scholar). To test whether this site could also bind chloride, we determined the Kd for chloride in the absence and in the presence of 20 mm NaN3 (Figs. 2B and 3A); we have also compared the affinity of CcO for azide with and without 50 mm NaCl (Fig. 4).Fig. 4Azide-induced shift of the g = 3 signal of oxidized CcO in the absence and presence of chloride.A, 40 μm oxidized oxidase (control (0)) and in the presence of 10 and 100 mm NaN3. B, 40 μm CcO with 50 mm NaCl present (0) and after addition of 10 and 100 mm NaN3. C, plots of normalized change in the difference EPR amplitudes of the g = 3 signal versus NaN3 concentration for CcO with and without 50 mm NaCl. Symbols, data; dashed lines, fits assuming one azide-binding site and saturation ∼80–90% at 100 mm NaN3. Conditions are the same as in Fig. 1.View Large Image Figure ViewerDownload Hi-res image Download (PPT) After the addition of 20 mm NaN3, the g = 3 signal of isolated CcO showed two closely positioned maxima (Fig. 3A), with a fraction of the signal unchanged and the remainder shifted downfield by about 35 G; this curve shape (as shown in Fig. 3) persists up to an azide concentration of 1 m. The fraction of CcO with an EPR spectrum almost unchanged from that of untreated enzyme could be interpreted as a population of enzyme that does not react with azide (Fig. 3A). That this is not the case was readily demonstrated. The addition of 10 mm sodium chloride to azide-free enzyme produced the expected upfield shift of the EPR spectrum (Fig. 3B), whereas addition of this same concentration of sodium chloride to the enzyme pretreated with sodium azide left the azide-modified EPR spectrum unchanged (Fig. 3A). The response of CcO to azide was again indicative of the presence of two populations of cytochrome oxidase, observed in the interaction of chloride with CcO and attributed to the modification of enzyme during isolation. As before, enzyme samples were collected at each stage of the purification and reacted with 200 mm azide. It was again observed that it was mainly chromatography on Sepharose Q that led to the development of this inhomogeneous response to ligand binding. It can be seen that the presence of azide decreases the affinity of CcO for chloride (Fig. 3). To estimate quantitatively the effect of azide, we again measured the high field shift of the g = 3 signal produced by raising the concentration of NaCl. Fitting the normalized plot of the signal increase on the high field side of the g = 3 peak resulted in a single binding site with a Kd of 70 mm (Fig. 2B). The same value was determined from a plot of the signal decrease at the low field side of the g = 3 peak. The result indicated that the two populations of CcO, characterized by the distinct maxima observed in the presence of 20 mm azide, showed the same affinity for chloride. This affinity was about 20-fold smaller than that determined in the absence of azide. At this point it would appear that our data can be explained in two ways: (i) there is only one ligand-binding site, and chloride and azide compete for it, or (ii) there are two different sites, but there is an interaction between them such that when azide occupies its specific site, the affinity of the second site for chloride is decreased 20-fold. To examine these alternatives we compared the affinity of CcO for azide in the presence and absence of 50 mm NaCl (Fig. 4). Surprisingly, at this concentration of NaCl, there was no reduction in the affinity of CcO for azide, as revealed in the spectra of enzyme treated with 10 mm azide and with and without 50 mm chloride (Fig. 4, A and B). In both cases, azide induced a low field shift of the g = 3 signal. Moreover, it appears the fraction of CcO that underwent the low field shift following reaction with 10 mm NaN3 was enha" @default.
• W2025531019 created "2016-06-24" @default.
• W2025531019 creator A5011278508 @default.
• W2025531019 creator A5051222931 @default.
• W2025531019 creator A5082769187 @default.
• W2025531019 date "2004-04-01" @default.
• W2025531019 modified "2023-09-30" @default.
• W2025531019 title "Two Sites of Interaction of Anions with Cytochrome a in Oxidized Bovine Cytochrome c Oxidase" @default.
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