Cyanide binding and active site structure in heme-copper oxidases: Normal coordinate analysis of iron-cyanide vibrations of CN− complexes of cytochromesba3 andaa3 (original) (raw)
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The Journal of biological chemistry, 1990
Cyanide binding to bovine heart cytochrome c oxidase at five redox levels has been investigated by use of infrared and visible-Soret spectra. A C-N stretch band permits identification of the metal ion to which the CN- is bound and the oxidation state of the metal. Non-intrinsic Cu, if present, is detected as a cyanide complex. Bands can be assigned to Cu+CN at 2093 cm-1, Cu2+CN at 2151 or 2165 cm-1, Fe3+CN at 2131 cm-1, and Fe2+CN at 2058 cm-1. Fe2+CN is found only when the enzyme is fully reduced whereas the reduced Cu+CN occurs in 2-, 3-, and 4-electron reduced species. A band for Fe3+CN is not found for the complex of fully oxidized enzyme but is for all partially reduced species. Cu2+CN occurs in both fully oxidized and 1-electron-reduced oxidase. CO displaces the CN- at Fe2+ to give a C-O band at 1963.5 cm-1 but does not displace the CN- at Cu+. Another metal site, noted by a band at 2042 cm-1, is accessible only in fully reduced enzyme and may represent Zn2+ or another Cu+. Bi...
The Journal of Physical Chemistry B, 2003
Resonance Raman and FTIR spectroscopies have been employed to investigate the structure of the heme a 3 sCtNsCu B complex of oxidized cytochrome aa 3 oxidase. The characterization of this complex is essential since a central issue in the physiological function of cytochrome oxidase is the extent to which the partially reduced dioxygen substrate interacts with the two metals. The resonance Raman spectra display two isotopesensitive vibrational modes at 488 and 406 cm -1 . The FTIR spectrum displays one isotope-sensitive mode at 2151 cm -1 . We assign the peak at 488 cm -1 to the FesCtNsCu B stretching mode, the peak at 406 cm -1 to the FesCtNsCu B bending mode, and the peak at 2151 cm -1 to the CtN stretching mode. The comparison between the data on CN-bound oxidized enzyme and the data on model compound 1 illustrates that changes in the both the CusNtC angle and N CN -Cu stretch will influence the overall frequency of Fe-CN, while a decrease in the Cu-N-C angle will decrease the triple bond character of bridging cyanide.
Biophysical Journal, 1981
ABSTRACr X-ray edge absorption of copper and extended fine structure studies of both copper and iron centers have been made of cytochrome oxidase from beef heart, Paracoccus dentrificans, and HB-8 thermophilic bacteria (1-2.5 mM in heme). The desired redox state (fully oxidized, reduced CO, mixed valence formate and CO) in the x-ray beam was controlled by low temperature (-1400C) and was continuously monitored by simultaneous optical spectroscopy and by electron paramagnetic resonance (EPR) monitoring every 30 min of x-ray exposure. The structure of the active site, a cytochrome a3-copper pair in fully oxidized and in mixed valence formate states where they are spin coupled, contains a sulphur bridge with three ligands 2.60 + 0.03 A from Fea3 and 2.18 ± 0.03 A from Cua3. The distance between Fea3 and Cua3 is 3.75 ± 0.05 A, making the sulphur bond angle 1030 reasonable for Sp3 sulphur bonding. The Fea3 first shell has four typical heme nitrogens (2.01 + 0.03 A) with a proximal nitrogen at 2.14 + 0.03 A. The sixth ligand is the bridging sulphur. The Cua3 first shell is identical to oxidized stellacyanin containing two nitrogens and a bridging sulphur. Upon reduction with CO, the active site is identical to reduced stellacyanin for the Cua3 first shell and contains the sulphur that forms the bridge in fully oxidized and mixed valence formate states. The Fea3 first shell is identical to oxyhemoglobin but has CO instead of 02. The other redox centers, Fea and the other "EPR detectable" Cu are not observed in higher shells of Fea3. Fea has six equidistant nitrogens and Cua has one (or two) nitrogens and three (or two) sulphurs with typical distances; these ligands change only slight on reduction. These structures afford the basis for an oxygen reduction mechanism involving oxy-and peroxy intermediates. 'a3 heme and Cua3 are identified with the active site of the oxidase, whereas Fea3 refers specifically to the iron atom of a3 heme. Correspondingly, a heme and Cu, refer to the electron reservoir component of the oxidase.
pH-Dependent Structural Changes at the Heme-Copper Binuclear Center of Cytochrome c Oxidase
Biophysical Journal, 2001
The resonance Raman spectra of the aa 3 cytochrome c oxidase from Rhodobacter sphaeroides reveal pH-dependent structural changes in the binuclear site at room temperature. The binuclear site, which is the catalytic center of the enzyme, possesses two conformations at neutral pH, assessed from their distinctly different Fe-CO stretching modes in the resonance Raman spectra of the CO complex of the fully reduced enzyme. The two conformations (␣ and ) interconvert reversibly in the pH 6 -9 range with a pKa of 7.4, consistent with Fourier transform infrared spectroscopy measurements done at cryogenic temperatures (D. M. Mitchell, J. P. Shapleigh, A. M. Archer, J. O. Alben, and R. B. Gennis, 1996, Biochemistry 35:9446 -9450). It is postulated that the different structures result from a change in the position of the Cu B atom with respect to the CO due to the presence of one or more ionizable groups in the vicinity of the binuclear center. The conserved tyrosine residue (Tyr-288 in R. sphaeroides, Tyr-244 in the bovine enzyme) that is adjacent to the oxygen-binding pocket or one of the histidines that coordinate Cu B are possible candidates. The existence of an equilibrium between the two conformers at physiological pH and room temperature suggests that the conformers may be functionally involved in enzymatic activity.
Journal of Biological Chemistry
Cyanide-binding to the heme-copper binuclear center of bo-type ubiquinol oxidase from Escherichia coli was investigated with Fourier transform-infrared and EPR spectroscopies. Upon treatment of the air-oxidized CNinhibited enzyme with excess sodium dithionite, a 12 C-14 N stretching vibration at 2146 cm ؊1 characteristic of the Fe O 3؉ -C؍N-Cu B 2؉ bridging structure was quickly replaced with another stretching mode at 2034.5 cm ؊1 derived from the Fe O 2؉ -C؍N moiety. The presence of ubiquinone-8 or ubiquinone-1 caused a gradual autoreduction of the metal center(s) of the air-oxidized CNinhibited enzyme and a concomitant appearance of a strong cyanide stretching band at 2169 cm ؊1 . This 2169 cm ؊1 species could not be retained with a membrane filter (molecular weight cutoff ؍ 10,000) and showed unusual cyanide isotope shifts and a D 2 O shift. These observations together with metal content analyses indicate that the 2169 cm ؊1 band is due to a Cu B ⅐CN complex released from the enzyme. The same species could be produced by anaerobic partial reduction of the CN-inhibited ubiquinol oxidase and, furthermore, of the CNinhibited cytochrome c oxidase; but not at all from the fully reduced CN-inhibited enzymes. These findings suggest that there is a common intermediate structure at the binuclear center of heme-copper respiratory enzymes in the partially reduced state from which the Cu B center can be easily released upon cyanide-binding.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1983
The midpoint redox potentials of Pseudomonas aeruginosa cytochrome c-551 and Rhodopseudomonas eiridis cytochrome c 2 were measured as a function of pH in the presence of Euglena cytochrome o558 and the results compared with those obtained in the presence of ferro-ferricyanide. The pattern of pH dependence observed for the two bacterial cytochromes was the same whether it was measured by equilibrium with another redox protein or with the inorganic redox couple. Thus, the pH dependence of redox potential is not a consequence of pH-dependent ligand binding. The midpoint potential of Ps. aeruginosa azurin was measured as a function of pH using both ferro-ferricyanide mixtures and redox equilibrium with horse cytochrome c or Rhodopseudomonas capsulata cytochrome c 2. In this case also the pattern of pH dependence obtained did not vary with the redox system used and it closely resembled that of Ps. aeruginosa cytochrome c-551. This is consistent with the observation that the equilibrium between cytochrome c-551 and azurin is relatively independent of pH. An equation was derived which described pH-dependent ligand binding and which can produce theoretical curves to fit the experimental pH dependence of redox potential for both cytochrome and azurin. However, the pronounced effect on such curves produced by varying the ligand association constants, and the insensitivity of the experimental data to changes in ionic strength, suggest that ligand binding effects do not account for the pH dependence of redox potential.
The EMBO Journal, 1992
The cupredoxin fold, a Greek key fl-barrel, is a common structural motif in a family of small blue copper proteins and a subdomain in many multicopper oxidases. Here we show that a cupredoxin domain is present in subunit II of cytochrome c and quinol oxidase complexes. In the former complex this subunit is thought to bind a copper centre called CUA which is missing from the latter complex. We have expressed the C-terminal fragment of the membrane-bound CyoA subunit of the Escherichia coli cytochrome o quinol oxidase as a water-soluble protein. Two mutants have been designed into the CyoA fragment. The optical spectrum shows that one mutant is similar to blue copper proteins. The second mutant has an optical spectrum and redox potential like the purple copper site in nitrous oxide reductase (N2OR). This site is closely related to CUA, which is the copper centre typical of cytochrome c oxidase. The electron paramagnetic resonance (EPR) spectra of both this mutant and the entire cytochrome o complex, into which the CUA site has been introduced, are similar to the EPR spectra of the native CUA site in cytochrome oxidase. These results give the first experimental evidence that CUA is bound to the subunit H of cytochrome c oxidase and open a new way to study this peculiar copper site.
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1994
Cytochrome aa3-600 is a terminal quinol oxidase of Bacillus subtilis, belonging to the large family of structurally and functionally related respiratory enzymes to which the mitochondrial cytochrome c oxidase also belongs. However, the Cu A center typical of the cytochrome c oxidases is lacking from cytochrome aa3-600. The presence of only one copper, viz. Cu B of the binuclear heme iron-copper site, makes cytochrome aa3-600 especially suitable for XAS analysis of this structure. Cu and Fe XAS data for fully oxidized cytochrome aa3-600 indicate a structure for the binuclear site similar to that previously reported for mitochondrial cytochrome c oxidase (see Biophys. J. 34, 465-468). Heme Fea3 has a proximal histidine nitrogen ligand 2.10 + 0.02 ,~ from the iron, and a distal S or C1 ligand at 2.36 ___ 0.03 A. The latter is also a ligand of Cu B (2.21 + 0.02 ,~), and apparently forms a bridge between the two metals which are 3.70 + 0.06 A apart. Cu B has two more close-lying ligands at 1.95 + 0.02 ,~, which are likely histidine nitrogens. The similarity between EXAFS of Cu B and type 1 'blue' copper is contrasted to EPR and optical spectroscopic properties of Cu B, and the nature of the bridging ligand is discussed.