Evidence for a band III analogue in the near-infrared absorption spectra of cytochrome c oxidase.

Ground state near-infrared absorption spectra of fully reduced unliganded and fully reduced CO (a2+ CuA+ a3(2+)-CO CuB+) cytochrome c oxidase were investigated. Flash-photolysis time-resolved absorption difference spectra of the mixed-valence (a3+ CuA2+ a3(2+)-CO CuB+) and the fully reduced CO complexes were also studied. A band near 785 nm (epsilon approximately 50 M-1cm-1) was observed in the fully reduced unliganded enzyme and the CO photoproducts. The time-resolved 785 nm band disappeared on the same timescale (t1/2 approximately 7 ms) as CO recombined with cytochrome a3(2+). This band, which is attributed to the unliganded five coordinate ferrous cytochrome a3(2+), has some characteristics of band III in deoxy-hemoglobin and deoxy-myoglobin. A second band was observed at approximately 710 nm (epsilon approximately 80 M-1cm-1) in the fully reduced unliganded and the fully reduced CO complexes. This band, which we assign to the low spin ferrous cytochrome a, appears to be affected by the ligation state at the cytochrome a3(2+) site.     

Characterisation of a near infra-red absorption band of the Escherichia coli quinol oxidase, cytochrome o, which is attributable to the high-spin ferrous haem of the binuclear site.

The bacterial quinol oxidase, cytochrome o, is an enzyme which is highly analogous to the better known cytochrome c oxidase, cytochrome aa3, but with the important difference that it lacks the near infra-red absorbing pigment CuA. In this article we report an absorption band in the near IR spectrum of cytochrome o with a maximal absorption at 758 nm, and which is attributable to the ferrous high-spin haem. The 758 nm band has an extinction coefficient of 0.2-0.3 mM-1.cm-1 at 758-800 nm. This region in cytochrome aa3 is dominated by the CuA absorption. The 758 nm absorption is lost on addition of CO or cyanide to the reduced enzyme. The carbon monoxide compound of cytochrome o also has absorbance bands in the near infra-red, and these may be attributable to a low-spin ferrous haem compound.     

Resonance Raman spectroscopy of cytochrome c oxidase and electron transport particles with excitation near the Soret band

We report the resonance Raman spectra of cytochrome $\text{c}$ oxidase, both solubilized and in electron transport particles using laser excitation near the Soret band. As in the spectra of other hemoproteins, such as cytochrome $\text{c}$, the shape and intensity of a number of bands change when the oxidation state is varied. However, one of the hemes of solubilized cytochrome $\text{c}$ oxidase shows redox behavior which is anomalous. Spectra of electron transport particles are dominated by cytochrome $\text{c}$ oxidase. There are, however, definite differences between spectra of solubilized cytochrome $\text{c}$ oxidase and electron transport particles in the oxidized states.     

Interaction of cytochrome c with cytochrome c oxidase. Photoaffinity labeling of beef heart cytochrome c oxidase with arylazido-cytochrome c.

Cytochrome c derivatives labeled with a 3-nitrophenylazido group at lysine 13, at lysine 22, or at both residues have been prepared. The interaction of the cytochrome c derivatives with beef heart cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) in the presence of ultrviolet light results in formation of a covalent complex between cytochrome c and the oxidase. Using the lysine 22 derivative, the polypeptide composition of the oxidase is not modified, nor is its catalytic activity, whereas with the lysine 13 derivative, the gel electrophoretic pattern is altered and the catalytic activity of the complex diminished. The data are consisten with a specfic covalent interaction of the lysine 13 derivative of cytochrome c with the polypeptide of molecular weight 23,700 (Subunit II) of cytochrome c oxidase.     

Cytochrome c binding affects the conformation of cytochrome a in cytochrome c oxidase.

Second derivative absorption spectroscopy has been used to assess the effects of complex formation between cytochrome c and cytochrome c oxidase on the conformation of the cytochrome a cofactor. When ferrocytochrome c is complexed to the cyanide-inhibited reduced or mixed valence enzyme, the conformation of ferrocytochrome a is affected. The second derivative spectrum of these enzyme forms displays two electronic transitions at 443 and 451 nm before complex formation, but only the 443-nm transition after cytochrome c is bound. This effect is not induced by poly-L-lysine, a homopolypeptide which is known to bind to the cytochrome c binding domain of cytochrome c oxidase. The effect is limited to cyanide-inhibited forms of the enzyme; no effect was observed for the fully reduced unliganded or fully reduced carbon monoxide-inhibited enzyme. The spectral signatures of these changes and the fact that they are exclusively associated with the cyanide-inhibited enzyme are both reminiscent of the effects of low pH on the conformation of cytochrome a (Ishibe, N., Lynch, S., and Copeland, R. A. (1991) J. Biol. Chem. 266, 23916-23920). These results are discussed in terms of possible mechanisms of communication between the cytochrome c binding site, cytochrome a, and the oxygen binding site within the cytochrome c oxidase molecule.     

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.     

The cytochrome c binding site on cytochrome c oxidase.

Cytochrome $\text{c}$ was chemically coupled to cytochrome $\text{c}$ oxidase using the reagent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) which couples amine groups to carboxyl residues. The products of this reaction were analyzed on 2.5–27% polyacrylamide gradient gels electrophoretically. Since cytochrome $\text{c}$ binds to cytochrome oxidase electrostatically in an attraction between certain of its lysine residues and carboxyl residues on the oxidase surface, EDC is an especially appropriate reagent probe for binding-subunit studies. Coupling of polylysine to cytochrome oxidase using EDC was also performed, and the products of this reaction indicate that polylysine, an inhibitor of the cytochrome $\text{c}$ reaction with oxidase, binds to the same oxidase subunit as does cytochrome $\text{c}$, subunit IV in the gel system used.     

The pH dependence of cytochrome a conformation in cytochrome c oxidase.

The pH dependence of the conformation of cytochrome a in bovine cytochrome c oxidase has been studied by second derivative absorption spectroscopy. At neutral pH, the second derivative spectra of the cyanide-inhibited fully reduced and mixed valence enzyme display two Soret electronic transitions, at 443 and 451 nm, associated with cytochrome a. As the pH is lowered these two bands collapse into a single transition at approximately 444 nm. pH titration of the cyanide-inhibited mixed valence enzyme suggests that the transition from the two-band to one-band spectrum obeys the Henderson Hasselbalch relationship for a single protonation event with a transition pKa of 6.6 +/- 0.1. No pH dependence is observed for the spectra of the fully reduced unliganded or CO-inhibited enzyme. Tryptophan fluorescence spectra of the enzyme indicate that no major disruption of protein structure occurs in the pH range 5.5-8.5 used in this study. Resonance Raman spectroscopy indicates that the cytochrome a3 chromophore remains in its ferric, cyanide-bound form in the mixed valence enzyme throughout the pH range used here. These data indicate that the transition observed by second derivative spectroscopy is not due simply to pH-induced protein denaturation or disruption of the cytochrome a3 iron-CN bond. The pH dependence observed here is in good agreement with those observed earlier for the midpoint reduction potential of cytochrome a and for the conformational transition associated with energy transduction in the proton pumping model of Malmstr?m (Malmstr?m, B. G. (1990) Arch. Biochem. Biophys. 280, 233-241). These results are discussed in terms of a model for allosteric communication between cytochrome a and the binuclear ligand binding center of the enzyme that is mediated by ionization of a single group within the protein.     

Properties of ubiquinol oxidase reconstituted from ubiquinol-cytochrome c reductase, cytochrome c and cytochrome c oxidase.

Ubiquinol-cytochrome c reductase (Complex III), cytochrome c and cytochrome c oxidase can be combined to reconstitute antimycin-sensitive ubiquinol oxidase activity. In 25 mM-acetate/Tris, pH 7.8, cytochrome c binds at high-affinity sites (KD = 0.1 microM) and low-affinity sites (KD approx. 10 microM). Quinol oxidase activity is 50% of maximal activity when cytochrome c is bound to only 25% of the high affinity sites. The other 50% of activity seems to be due to cytochrome c bound at low-affinity sites. Reconstitution in the presence of soya-bean phospholipids prevents aggregation of cytochrome c oxidase and gives rise to much higher rates of quinol oxidase. The cytochrome c dependence was unaltered. Antimycin curves have the same shape regardless of lipid/protein ratio, Complex III/cytochrome c oxidase ratio or cytochrome c concentration. Proposals on the nature of the interaction between Complex III, cytochrome c and cytochrome c oxidase are considered in the light of these results.     

Zinc cytochrome c fluorescence as a probe for conformational changes in cytochrome c oxidase.

Zinc cytochrome c forms tight 1:1 complexes with a variety of derivatives of cytochrome c oxidase. On complex-formation the fluorescence of zinc cytochrome c is diminished. Titrations of zinc cytochrome c with cytochrome c oxidase, followed through the fluorescence emission of the former, have yielded both binding constants (K approximately 7 x 10(6) M-1 for the fully oxidized and 2 x 10(7) M-1 for the fully reduced enzyme) and distance information. Comparison of steady-state measurements obtained by absorbance and fluorescence spectroscopy in the presence and in the absence of cyanide show that it is the reduction of cytochrome a and/or CuA that triggers a conformational change: this increases the zinc cytochrome c to acceptor (most probably cytochrome a itself) distance by some 0.5 nm. Ligand binding to the fully oxidized or fully reduced enzyme leaves the extent of fluorescence quenching unchanged, whereas binding of cyanide to the half-reduced enzyme (a2+CuA+CuB2+-CN(-)-a3(3+)) enhances fluorescence emission relative to that for the fully reduced enzyme, implying further relative movement of donor and acceptor.     

Evidence for a ferryl Fea3 in oxygenated cytochrome c oxidase

Evidence is reported which shows that a reactive ferryl Fea3/cupric CuB binuclear couple is present at the dioxygen reduction site in "oxygenated" cytochrome c oxidase; when the fully reduced enzyme is reoxidized at low temperatures; and when partially reduced cytochrome c oxidase is reoxidized with dioxygen at room temperature.     

Thermodynamic volume cycles for electron transfer in the cytochrome c oxidase and for the binding of cytochrome c to cytochrome c oxidase.

Dilatometry is a sensitive technique for measuring volume changes occurring during a chemical reaction. We applied it to the reduction-oxidation cycle of cytochrome c oxidase, and to the binding of cytochrome c to the oxidase. We measured the volume changes that occur during the interconversion of oxidase intermediates. The numerical values of these volume changes have allowed the construction of a thermodynamic cycle that includes many of the redox intermediates. The system volume for each of the intermediates is different. We suggest that these differences arise by two mechanisms that are not mutually exclusive: intermediates in the catalytic cycle could be hydrated to different extents, and/or small voids in the protein could open and close. Based on our experience with osmotic stress, we believe that at least a portion of the volume changes represent the obligatory movement of solvent into and out of the oxidase during the combined electron and proton transfer process. The volume changes associated with the binding of cytochrome c to cytochrome c oxidase have been studied as a function of the redox state of the two proteins. The volume changes determined by dilatometry are large and negative. The data indicate quite clearly that there are structural alterations in the two proteins that occur on complex formation.     

The second derivative electronic absorption spectrum of cytochrome c oxidase in the Soret region.

The electronic absorption spectrum of solubilized beef heart cytochrome c oxidase was analyzed in the 400-500 nm region to identify the origin of doublet features appearing in the second derivative spectrum associated with ferrocytochrome a. This doublet, centered near 22,600 cm(-1), was observed in the direct absorption spectrum of the a(2+)a(3)(3+).HCOO(-) form of the enzyme at cryogenic temperatures. Since evidence for this doublet at room temperature is obtained only on the basis of the second derivative spectrum, a novel mathematical approach was developed to analyze the resolving power of second derivative spectroscopy as a function of parameterization of spectral data. Within the mathematical limits defined for resolving spectral features, it was demonstrated that the integrated intensity of the doublet feature near 450 nm associated with ferrocytochrome a is independent of the ligand and oxidation state of cytochrome a(3). Furthermore, the doublet features, also observed in cytochrome c oxidase from Paracoccus denitrificans, were similarly associated with the heme A component and were correspondingly independent of the ligand and oxidation state of the heme A(3) chromophore. The doublet features are attributed to lifting of the degeneracy of the x and y polarized components of the B state of the heme A chromophore associated with the Soret transition.