The nature of the copper atoms of cytochrome c oxidase as studied by optical and x-ray absorption edge spectroscopy.

X-ray absorption edge spectroscopy has been used to study the copper of 1--2 mM cytochrome c oxidase in the resting oxidized, mixed-valence, and fully reduced states. A comparison was made of this protein with copper complexes and with natural and artificial copper proteins. Spectra were obtained with synchrotron radiation from the SPEAR storage ring using highly sensitive fluorescence detectors. Temperatures of -80 to -120 degrees C were employed further to improve the stability of the samples and to avoid the possibility of either auto- or photon-induced reduction of the materials, which might have occurred in previous studies. In order to characterize the valence states of the Cu and Fe components, the samples were monitored by infrared and visible spectroscopy before and after irradiation by the X-ray beam. The combination of the optical and X-ray absorption techniques has afforded a deconvolution of the four species of copper in the various states of cytochrome c oxidase and the tentative assignment of Cu alpha, the copper redox coupled to the heme alpha of cytochrome alpha, as a highly covalent type of copper and Cu alpha 3, the copper of cytochrome alpha 3, as a more ionic 'blue' type I copper. The implications of these findings upon the mechanism of action of cytochrome oxidase are briefly outlined.     

Structure and reactivity of multiple forms of cytochrome oxidase as evaluated by X-ray absorption spectroscopy and kinetics of cyanide binding

The extended X-ray absorption fine structure (EXAFS) data show differences between the active site structures of different cytochrome oxidase preparations. In the resting (as isolated) state of the Yonetani preparation, the bridging atom between Fe3+a3 and Cu2+a3 is present [Powers, L., Chance, B., Ching, Y., & Angiolillo, P. (1981) Biophys. J. 34, 465], whereas in another preparation (e.g., Hartzell-Beinert), this atom seems to be bound only to Fe3+a3 in a significant fraction of the molecules. Both preparations bind cyanide in a multiphasic fashion, suggesting that the resting cytochrome oxidase is not homogeneous but rather is a mixture of several forms. The proportion of these forms as detected by cyanide binding kinetics differs for different preparations. However, upon reduction and reoxidation (conversion to the "oxygenated" form) the cyanide binding kinetics become monophasic and all preparations of the oxygenated form bind cyanide at the same rate. Thus, a combination of structural and kinetic approaches seems necessary for evaluation of the nature of the active site of cytochrome oxidase in its various forms.     

Protolytic reactions on reduction of cytochrome c oxidase studied by ATR-FTIR spectroscopy

Reduction of cytochrome c oxidase is coupled to proton uptake, and the reduced-minus-oxidized FTIR spectrum should include signatures of protonation of protolytic centers. The major part of the spectrum shows only small differences between acidic and alkaline conditions, which is consistent with the rather weak pH dependence of the proton uptake stoichiometry. Here we aim at revealing redox state-dependent protonatable sites and present a comprehensive investigation over a wide pH range. The reduced-minus-oxidized transition of cytochrome c oxidase from Paracoccus denitrificans was studied by means of Fourier transform infrared spectroscopy in the pH range 5.2-9.5. Effects of pH were analyzed as the difference between reduced-minus-oxidized FTIR spectra at different pH values. Two pH-dependent processes with apparent pKa values of 6.6 and 8.4 and Hill coefficients 0.9 and 0.1, respectively, were found by this methodology. A sharp OH band appears in the IR "water region" on reduction of the enzyme, independent of pH in the range 6.5-9.0, and downshifted by approximately 940 cm-1 on changing the solvent to D2O and by 10 cm-1 on H216O/H218O isotope exchange. This feature of an asymmetric water molecule may belong to water that is produced in the binuclear center upon reduction or to a structured water molecule that loses a hydrogen bond.     

Novel structural variation of silver(I)-pyridine complexes in nitromethane as studied by X-ray absorption spectroscopy

The XAFS spectra were measured at around the Ag K-edge of the Ag(I) ion in nitromethane (NM) with a variety of concentrations of pyridine (PY). In NM without PY, the Ag(I) ion is tetrahedrally solvated by four NM molecules similar to those in most solvents. The Ag-O bond length in NM solvent is longer than that in aqueous solution, indicating the low donating ability of NM. The mono-, bis-, tris-, and tetrakis-pyridine complexes are formed in NM by the addition of PY. The EXAFS analyses reveal that the structure of the formed PY complex in NM is linear for Ag(py)(nm)⁺, linear for , triangular for , and tetrahedral for . The longer Ag-O bond length for Ag(py)(nm)⁺ than that for and the release of bound NM molecules at the formation of Ag(py)(nm)⁺ are interpreted to be due to the strong σ donating property of PY. The Ag-N bond length (220 pm) for is intermediate between 216 pm for and 228 pm for . The formation equilibria of and are analyzed on the basis of the changeover of EXAFS spectra as a function of the total concentrations of Ag⁺ and PY in NM.     

Plutonium(III) complexation by humic substances studied by X-ray absorption fine structure spectroscopy

We determined structural parameters for the near-neighbor surrounding of plutonium(III) in complexes with humic and fulvic acids at pH 1 and for the purpose of comparison also for the plutonium(III) aquo ion by means of X-ray absorption fine structure (XAFS) spectroscopy. It could be shown that in the complexes with humic substances as well as in the plutonium(III) aquo ion sample the trivalent oxidation state of plutonium was stable within the time of the experiment. In the humate and fulvate complexes, the plutonium(III) is surrounded by about eight oxygen atoms with an average Pu–O distance of 2.48 ± 0.02 Å. The structural parameters determined for plutonium(III)–humate and –fulvate complexes were compared to structural parameters of plutonium(III) and plutonium(IV) aquo ions.     

Co-rebinding in myoglobin as seen by time-resolved X-ray absorption spectroscopy

The dynamics of selected conformational coordinates, key roles in the understanding of the CO-rebinding process, are investigated in horse heart carbonmonoxy myoglobin (MbCO) through time-resolved X-ray absorption spectroscopy. We present here the results obtained at 90 K in the second time scale. The approach of the CO molecule towards the Fe atom in the active site pocket is speculated to act as a natural precursor to the Fe displacement with the consequent undoming of the protein porphyrin plane. The arrangement of the Fe-C-O bonding angle geometry follows and the final MbCO active site configuration is completely reached within 1 min.     

Alkaline isomerization of thermoresistant cytochrome c-552 and horse heart cytochrome c studied by absorption and resonance raman spectroscopy

The structure of the thermoresistant cytochrome c (552, Thermus thermophilus) has been investigated at neutral and alkaline pH by absorption and resonance Raman spectroscopy and compared with that of horse heart cytochrome c. The ligands of the ferricytochrome c-552 at neutral pH are considered to be histidine and methionine, whereas the ligands of ferrocytochrome c-552 are histidine and another nitrogen base, histidine or lysine. Ferric cytochrome c-552 undergoes an alkaline isomerization with a pK of 12.3 (25°C), accompanied by a ligand exchange. Horse heart cytochrome c has at least three isomerization states at alkaline pH (pK 9.3, 12.9 and >13.5 at 25°C). The replacement of the sixth ligand may not be involved in the second isomerization.The thermodynamic parameters for the isomerization were also estimated. The entropy change upon isomerization of cytochrome c-552 is negative, whereas for that of horse heart cytochrome c the entropy change is positive.     

Complexation of cadmium in seeds and vegetative tissues of the cadmium hyperaccumulator Thlaspi praecox as studied by X-ray absorption spectroscopy

The cadmium hyperaccumulator Thlaspi praecox Wulfen (Brassicaceae) can accumulate unusually high amounts of Cd (>1,000 μg g^?1 dry weight) in its seeds without drastically affecting seed viability. As embryonic tissues are the most sensitive to Cd toxicity, the aim of this study was to investigate the Cd coordination and ligand environment in seeds of field collected T. praecox using extended X-ray absorption fine structure (EXAFS), and to compare the Cd ligand environment to that in the vegetative tissues of the plant. In intact seeds and isolated embryos, almost two thirds of the Cd ligands were thiol groups (Cd-S-C-). In addition, there was coordination to phosphate groups via bridging oxygens (Cd-O-P-), as for phytate, although this ligand was not observed in the vegetative organs and tissues. In roots and shoots up to 80% of the Cd ligands were oxygen ligands that are provided by the cell walls and by organic acids stored in vacuoles. In leaf epidermis only a slightly higher percentage of oxygen ligands was detected, as compared to the mesophyll, making vacuolar compartmentation and binding to the cell walls the main detoxification mechanisms in both of these leaf tissues.     

Active site conformation in myoglobin as determined by X-ray absorption spectroscopy

X-ray absorption fine structure experiments were performed to study structural and dynamic aspects of the active site of various forms of myoglobin. The structures determined for deoxyMb, MbCO, and MbO2 are consistent with the structure established by X-ray absorption fine structure experiment and X-ray crystallography. The first shell of ferrous MbNO determined contains 5 nitrogens located at 2.02 A and a short NO bond length of 1.76 A. This study focuses on the change of the XAFS Debye-Waller factor with temperature, which is a measure of thermal and static disorder. It was found that the changes of Debye-Waller factor with temperature for the Mb proteins, except deoxyMb, are consistent with a simple Einstein model, in which a single frequency was assumed for the bond stretching modes. In contrast, the temperature dependence of deoxyMb cannot be fitted to the Einstein model and a large disorder was found at low temperatures, which indicates the existence of conformational substates of the active site.     

Alkaline isomerization of thermoresistant cytochrome c-552 and horse heart cytochrome c studied by absorption and resonance Raman spectroscopy.

The structure of the thermoresistant cytochrome c (552, Thermus thermophilus) has been investigated at neutral and alkaline pH by absorption and resonance Raman spectroscopy and compared with that of horse heart cytochrome c. The ligands of the ferricytochrome c-552 at neutral pH are considered to be histidine and methionine, whereas the ligands of ferrocytochrome c-552 are histidine and another nitrogen base, histidine or lysine. Ferric cytochrome c-552 undergoes an alkaline isomerization with a pK of 12.3 (25 degrees C), accompanied by a ligand exchange. Horse heart cytochrome c has at least three isomerization states at alkaline pH (pK 9.3, 12.9 and greater than 13.5 at 25 degrees C). The replacement of the sixth ligand may not be involved in the second isomerization. The thermodynamic parameters for the isomerization were also estimated. The entropy change upon isomerization of cytochrome c-552 is negative, whereas for that of horse heart cytochrome c the entropy change is positive.     

Cytochrome c and cytochrome c peroxidase complex as studied by resonance Raman spectroscopy.

Complex formation between ferricytochrome c peroxidase (CCP) and ferricytochrome c from yeast [cyt(Y)] and horse heart [cyt(H)] was studied by resonance Raman spectroscopy. On the basis of a detailed spectral analysis of the free proteins, it was possible to attribute changes in the spectra of the complexes to the individual proteins. At pH 7.0 both cyt(Y) and cyt(H) binding induces an increase in the six-coordinate low-spin configuration of CCP from 9% to 19% at the expense of the five-coordinate high-spin state, which drops from 84% to 74%. In the free and complexed state, CCP exhibits a constant fraction of the six-coordinate high-spin form (approximately 7%). In addition to affecting the coordination state, there is also a cyt-specific structural response of CCP to complexation. In the cyt(Y)-CCP complex, the peripheral vinyl and propionate substituents of CCP are more rigidly fixed in the protein matrix, whereas binding of cyt(H) only slightly perturbs the conformations of these side chains. The biological significance of the conformational changes in CCP are discussed. In contrast to CCP, there are no detectable structural changes in either cyt(Y) or cyt(H) upon complex formation.     

Resonance Raman spectroscopy and enhanced photoreducibility for the 420 nm pulsed form of cytochrome oxidase

Resonance Raman (RR) spectra, with 413.1 nm Kr+ laser excitation, are reported for cytochrome oxidase in resting, reduced, and 428 nm (oxygenated) forms, and for the first time, in the 420 nm (pulsed) forms [(1984) J. Biol. Chem. 259, 2073-2076]. The differences between the resting, 420 nm, and 428 nm forms' RR spectra are small. All these forms contain FeIII only, as indicated by single v4 bands at approximately 1371 cm-1, and the reoxidized forms show partial conversion from high- to intermediate- or low-spin heme a3 (intensity shift from 1575 to 1588 cm-1 for v2). The 420 nm form differs strikingly from both the 428 nm and resting forms, however, in being much more readily photoreduced by the laser illumination. This property is linked to the protein conformational change believed to be responsible for the greater accessibility to exogenous ligands of the heme a3 in the 420 nm form.     

UV optical absorption by protein radicals in cytochrome c oxidase

The UV properties of key oxygen intermediates of cytochrome c oxidase have been investigated by transient absorption spectroscopy. The temporal behavior of P(m) species upon aerobic incubation with CO or in the reaction with H(2)O(2) is closely concurred by a new optical shift at 290/260 nm. In the acid-induced conversion of P(m) to F(*), it is replaced by another shift at 323/288 nm. The wavelength and intensity of the UV signal observed in F(*) match closely the properties of model Trp? in agreement with results of ENDOR studies on this species. The UV spectrum of Tyr* gives the closest match with the 290/260 nm signal observed in P(m). On the basis of analysis of possible UV chromophores in CcO and similarity to Tyr*, the 290/260 nm signal is proposed to originate from the H(240)-Y(244)* site. Possible effects of local environment on UV properties of this site are discussed.