pH dependence of the tryptophan fluorescence in cytochrome c oxidase: further evidence for a redox-linked conformational change associated with CuA

When the low-potential metal centers of cytochrome c oxidase are reduced, the enzyme undergoes a conformational transition that shifts the fluorescence maximum of the emitting tryptophan residues from 329 to 345 nm. At pH 7.4, the change in this tryptophan fluorescence intensity is a nonlinear function of the electron equivalents added to the cyanide-inhibited enzyme. This nonlinear behavior is a result of the difference in redox potential between cytochrome a and CuA, which, at equilibrium, favors electron occupancy at cytochrome a. Studies on the cyanide-inhibited enzyme suggest that the conformational change is associated with reduction of CuA [Copeland, R. A., Smith, P. A., & Chan, S. I. (1987) Biochemistry 26, 7311-7316]. In this work we present tryptophan fluorescence data for the cyanide-inhibited enzyme at pH 8.9. Because of the pH dependence of the midpoint potential of cytochrome a in this form of the enzyme, the two low-potential centers become virtually isopotential at pH 8.9. The results obtained confirm our earlier conclusion that the observed conformational change is linked to the reduction of CuA only, rather than to the redox activity of both low-potential metal centers. We find that, in partially reduced cyanide-inhibited oxidase, raising the pH from 7.4 to 8.9 results in an intensification and red shift of the enzyme's tryptophan emission as the electron occupancy redistributes from cytochrome a to CuA. Moreover, when the fluorescence change is plotted as a function of the number of electrons added to the enzyme at pH 8.9, the data fit the nearly linear function expected for a conformational change triggered by reduction of CuA exclusively.     

Redox-linked conformational changes in bovine heart cytochrome c oxidase: picosecond time-resolved fluorescence studies of cyanide complex

Picosecond time-resolved fluorescence studies are carried out on cyanide-inhibited and heat-modified cytochrome c oxidase in aqueous lauryl maltoside surfactant solution, as well as in an aqueous vesicle, to understand the conformational changes associated with electron transfer and proton pumping activity of the enzyme. The tryptophan fluorescence decay profiles follow a four exponential model, which also matches the lifetime maxima obtained in a maximum entropy method analysis. The fast lifetime components are highly affected by the reduction and chemical modification of the enzyme. Changes in these lifetime components are related to the conformational changes in the vicinity of the heme centers of the enzyme. The cyanide-inhibited enzyme in the oxidized form shows a fluorescence decay profile similar to that of the native oxidized form, indicating that the conformational changes due to cyanide binding are very small. However, reduction of the cyanide-inhibited enzyme that leaves cyanide bound heme alpha3 oxidized causes a large increase in the fluorescence lifetimes, which indicates very significant conformational changes due to electron transfer to the dinuclear Cu(A) and heme alpha centers. A comparison of the tryptophan fluorescence decay of various other modified forms of the enzyme leads us to propose that the possible site of conformational coupling is located near heme alpha instead of the binuclear heme alpha3-Cu(B) center.     

Conversion of CuA to a type II copper in cytochrome c oxidase

When cytochrome c oxidase is incubated at 43 degrees C for approximately 75 min in a solution containing the zwitterionic detergent sulfobetaine 12, the CuA site is converted into a type II copper as judged by changes in the 830-nm absorption band and the EPR spectrum of the enzyme. SDS-PAGE and sucrose gradient ultracentrifugation indicate concomitant loss of subunit III and monomerization of the enzyme during the heat treatment. Comparison of the optical and resonance Raman spectra of the heat-treated and native protein shows that the heme chromophores are not significantly perturbed; the resonance Raman data indicate that the small heme perturbations observed are limited to the cytochrome a3 site. Proton pumping measurements, conducted on the modified enzyme reconstituted into phospholipid vesicles, indicate that these vesicles are unusually permeable toward protons during turnover, as previously reported for the p-(hydroxymercuri)benzoate-modified oxidase and the modified enzyme obtained by heat treatment in lauryl maltoside. The sulfobetaine 12 modified enzyme is no longer capable of undergoing the recently reported conformational transition in which the tryptophan fluorescence changes upon reduction of the low-potential metal centers. Control studies on the monomeric and subunit III dissociated enzymes suggest that the disruption of this conformational change in the heat-treated oxidase is most likely associated with perturbation of the CuA site. These results lend support to the suggestion that the fluorescence-monitored conformational change of the native enzyme is initiated by reduction of the CuA site [Copeland et al. (1987) Biochemistry 26, 7311].     

Characterization of reductant-induced, tryptophan fluorescence changes in cytochrome oxidase

We have measured the steady-state tryptophan fluorescence spectrum of cytochrome oxidase in its oxidized and fully reduced states. Reduction of the oxidized enzyme by sodium dithionite causes an apparent shift in the fluorescence emission maximum from 328 nm, in the oxidized enzyme, to 348 nm, in the reduced enzyme. This spectroscopic change has been observed previously and assigned to a redox-linked, conformational change in cytochrome oxidase [Copeland, R. A., Smith, P. A., & Chan, S. I. (1987) Biochemistry 26, 7311-7316]. When dithionite-reduced enzyme sits in an open cuvette, the enzyme returns to the oxidized state, and the fluorescence maximum shifts back to 328 nm. However, the time course of the fluorescence change does not follow the redox state of the enzyme, monitored spectrophotometrically at 445,605, and 820 nm, but follows the disappearance of dithionite, which absorbs at 315 nm. Moreover, when the fluorescence emission spectrum of the dithionite-reduced enzyme is corrected for the absorbance due to dithionite, the fluorescence maximum is found 2 nm blue shifted, relative to that of the oxidized enzyme, at 326 nm. This dithionite-induced, red-shifted steady-state tryptophan fluorescence is also seen with the non-heme-containing enzyme carboxypeptidase A. The tryptophan emission spectrum of untreated carboxypeptidase A is at 332 nm, whereas in the presence of dithionite the emission spectrum of carboxypeptidase A is at 350 nm. When corrected for the absorbance of dithionite, the tryptophan emission maximum is at 332 nm. We have also used the photoreductant 3,10-dimethyl-5-deazaisoalloxazine (deazaflavin) to reduce cytochrome oxidase.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Identification of cytochrome a and a3 in yeast cells.

1) Cells of Saccharomyces cerevisiae have been analysed by single and double-bean spectroscopy. Evidence is given for two components of cytochrome c oxidase in the alpha-region of their absorption spectrum. A rapidly reduceable component with a maximum at 600 nm and a slowly reduceable component with a maximum at 604 nm contribute about equal amounts to the total alpha-absorption of cytochrome c oxidase. 2) The component absorbing at 600 nm was identified as the high-potential component with a redox potential of 340 - 355mV, and the 604-nm component as the low-potential component of cytochrome c oxidase with redox potential of 180 - 190 mV. 3) Both components can be characterized by analysing the reduction kinetics in the presence of carbon monoxide. In the presence of saturating concentrations of carbon monoxide, an oxygen pulse leads to a rapid oxidation and subsequent reduction of cytochrome c oxidase, but the rapid reduction phase at 600 nm completely disappears, demonstrating its identity with cytochrome a3, which, being liganded by carbon monoxide in its reduced state, cannot react any more. The component which becomes oxidized and later reduced in the presence of carbon monoxide -- by definition cytochrome a -- has an absorption maximum at 604 nm. 4) The total extinction change at 604 nm in the presence of carbon monoxide is nearly as high as in its absence, but the reduction occurs in two phases and only the second phase, which contributes 50 - 60% to the total absorbance, corresponds in redox potential and kinetic properties to cytochrome a. Because the redox potential of the first reduction phase is very close to that of the low-potential copper atom of cytochrome c oxidase, it is concluded that the apparent increase in the extinction coefficient of cytochrome a in the presence of carbon monoxide is the result of a strong interaction between the ligand fields of cytochrome a and copper, induced by the binding of carbon monoxide to reduced cytochrome a3.     

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.     

Reduction of CuA induces a conformational change in cytochrome c oxidase from Paracoccus denitrificans.

Cytochrome c oxidase (cytochrome aa3) from Paracoccus denitrificans contains a tightly bound manganese(II) ion, which responds to reduction of the enzyme by a change in its EPR signal (Seelig et al. (1981) Biochim. Biophys. Acta 636, 162-167). In this paper, the nature of this phenomenon is studied and the bound manganese is used as a reporter group to monitor a redox-linked conformational change in the protein. A reductive titration of the cyanide-inhibited enzyme shows that the change in the manganese EPR signal is associated with reduction of CuA. The change appears to reflect a rearrangement in the rhombic octahedral coordination environment of the central Mn2+ atom and is indicative of a redox-linked conformational transition in the enzyme. The manganese is likely to reside at the interface of subunits I and II, near the periplasmic side of the membrane. One of its ligands may be provided by the transmembrane segment X of subunit I, which has been suggested to contribute ligands to cytochrome a and CuB as well. Another manganese ligand is a water oxygen, as indicated by broadening of the manganese EPR signal in the presence of H2(17)O.     

Detection, characterization, and quenching of the intrinsic fluorescence of bovine heart cytochrome c oxidase

The intrinsic fluorescence of lauryl maltoside solubilized bovine heart cytochrome c oxidase has been determined to arise from tryptophan residues of the oxidase complex. The magnitude of the fluorescence is approximately 34% of that from n-acetyltryptophanamide (NATA). This level of fluorescence is consistent with an average heme to tryptophan distance of 30 A. The majority of the fluorescent tryptophan residues are in a hydrophobic environment as indicated by the fluorescence emission maximum at 328 nm and the differing effectiveness of the quenching agents: Cs+, I-, and acrylamide. Cesium was ineffective up to a concentration of 0.7 M, whereas quenching by the other surface quenching agent, iodide, was complex. Below 0.2 M, KI was ineffective whereas between 0.2 and 0.7 M 15% of the tryptophan fluorescence was found to be accessible to iodide. This pattern indicates that protein structural changes were induced by iodide and may be related to the chaotropic character of KI. Acrylamide was moderately effective as a quenching agent of the oxidase fluorescence with a Stern-Volmer constant of 2 M-1 compared with acrylamide quenching of NATA and the water-soluble enzyme aldolase having Stern-Volmer constants of 12 M-1 and 0.3 M-1, respectively. There was no effect of cytochrome c on the tryptophan emission intensity from cytochrome c oxidase under conditions where the two proteins form a tight, 1:1 complex, implying that the tryptophan residues near the cytochrome c binding site are already quenched by energy transfer to the homes of the oxidase. The lauryl maltoside concentration used to solubilize the enzyme did not affect the fluorescence of NATA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hexaammineruthenium as an electron donor to mitochondrial cytochrome oxidase: membrane potential generation in the absence of cytochrome c

Cytochrome c oxidase can generate membrane potential in the absence of cytochrome c (e.g., in cytochrome c-deficient mitochondria or in proteoliposomes) with hexaammineruthenium as an artificial electron donor. Of several other redox mediators tested, phenazine methosulfate was found to be an efficient artificial substrate for membrane energization by cytochrome oxidase, whereas TMPD, DAD, DCPIP or ferrocyanide are virtually ineffective. The ability of Ru(NH3)6(2+) and phenazine methosulfate to support the generation of delta psi by cytochrome c-oxidase correlates with their effectiveness as electron donors to cytochrome a in the cyanide-inhibited membrane-bound enzyme.     

Analysis of inhibitor binding to the mitochondrial cytochrome c reductase by fluorescence quench titration. Evidence for a 'catalytic switch' at the Qo center

The binding characteristics of inhibitors of the mitochondrial cytochrome c reductase were studied by fluorescence quench titration. Based on the standard binding equation, the applied numerical method allowed the online recorded titration curves to be interpreted by fitting the Kd, the number of binding sites, and the specific fluorescence of the free and the bound inhibitor. For the Qi center, 2-n-nonyl-4-hydroxyquinoline N-oxide and for the Qo center (E)-beta-methoxyacrylate-stilbene (MOA-stilbene) were used as fluorescing inhibitors. The experiments could be extended to other, non-fluorescing inhibitors by competition analysis. Using this method we were able to compare the binding behaviour of Qi and Qo center inhibitors under different redox states of the enzyme using the same experimental set up. We studied the competition between inhibitors of the cytochrome c reductase representative for all subgroups and demonstrated that at least three inhibitor binding sites exist, two located in the Qo center, one located in the Qi center. Determination of the dissociation constants of the oxidized, the partially reduced and the fully reduced enzyme showed that inhibitor binding at the Qi center is not redox-dependent. In contrast, the binding of MOA-stilbene to the Qo center is decreased after reduction of the iron-sulfur center and cytochrome c1, whereas this redox change increases the affinity for a Qo center inhibitor of the hydroxynaphthoquinone type, 3-n-undecyl-2-hydroxynaphthoquinone. From these results, aware of the fact that the inhibitory mechanism at the Qo center is a non-competitive one, we made the hypothesis of a 'catalytic switch' to explain both the bifurcation of electron flow and the inhibition at the Qo center. A steric blockage of one of two conformational states could serve as a cogent explanation for the great structural variability of the inhibitors and differential effects on the redox centers exerted by the inhibitors. Moreover, the proposed 'switch' gives some insight into other experimental results which are difficult to explain with the ubiquinone cycle as currently formulated.     

Protein and lipid structural transitions in cytochrome c oxidase-dimyristoylphosphatidylcholine reconstitutions

The thermotropic behavior of the mitochondrial enzyme cytochrome c oxidase (EC 1.9.3.1) reconstituted in dimyristoylphosphatidylcholine (DMPC) vesicles has been studied by using high-sensitivity differential scanning calorimetry and fluorescence spectroscopy. The incorporation of cytochrome c oxidase into the phospholipid bilayer perturbs the thermodynamic parameters associated with the lipid phase transition in a manner analogous to other integral membrane proteins: it reduces the enthalpy change, lowers the transition temperature, and reduces the cooperative behavior of the phospholipid molecules. Analysis of the dependence of the enthalpy change on the protein:lipid molar ratio indicates that cytochrome c oxidase prevents 99 +/- 5 lipid molecules from participating in the main gel-liquid-crystalline transition. These phospholipid molecules presumably remain in the same physical state below and above the transition temperature of the bulk lipid, thus providing a more or less constant microenvironment to the protein molecule. The effect of the phospholipid bilayer matrix on the thermodynamic stability of the cytochrome c oxidase complex was examined by high-sensitivity differential scanning calorimetry. Detergent (Tween 80)-solubilized cytochrome c oxidase undergoes a complex, irreversible thermal denaturation process centered at 56 degrees C and characterized by an enthalpy change of 550 +/- 50 kcal/mol of enzyme complex. Reconstitution of the cytochrome c oxidase complex into DMPC vesicles shifts the transition temperature upward to 63 degrees C, indicating that the phospholipid bilayer moiety stabilizes the native conformation of the enzyme. The lipid bilayer environment contributes approximately 10 kcal/mol to the free energy of stabilization of the enzyme complex. The thermal unfolding of cytochrome c oxidase is not a two-state process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pressure-induced effects on cytochrome oxidase: the aerobic steady state

If cytochrome c oxidase is subjected to pressure during the aerobic steady state, large spectral changes are apparent. These seem to be associated with the inhibition of electron transport within the oxidase. The volume change for the transition is about 80 mL/mol. When the oxidase in the aerobic steady state, with porphyrin cytochrome c (the iron-free derivative of cytochrome c) bound to it, is subjected to pressure, the porphyrin derivative is released. This results from a change in the dissociation constant of the complex. Whereas the dissociation constant during turnover is about 1.25 X 10(-8) M, during pressure-induced inhibition the dissociation constant appears to be about an order of magnitude greater. It appears as though the binding site of the inhibited, partially reduced enzyme more closely resembles that of the fully reduced enzyme than that of the enzyme during the aerobic steady state.     

Interaction of the extrinsic potential-sensitive molecular probe diS-C3-(5) with pigeon heart mitochondria under equilibrium and time-resolved conditions

Some aspects of the interaction of the extrinsic, potential-sensitive, molecular probe diS-C3-(5) with pigeon heart mitochondria are reported in this paper. Binding studies based on fluorimetry indicate that the ratio of the dissociation constant to the maximum number of binding sites, KD/n, is larger for succinate-containing mitochondria than that for cyanide-inhibited preparations. These observations suggest that the basis of the energy-dependent diS-C3-(5) optical signals is the ejection of the probe from the mitochondrial membrane. A more detailed analysis indicated that the major change in the binding parameters is a reduction in the maximum number of binding sites, n, when a charge gradient is formed at the expense of substrate. Using rapid mixing techniques, the time course of the passive association of diS-C3-(5) with mitochondria, that of the glutamate- and ATP-dependent optical signals, and the effect of this probe on the rate at which the energy-dependent cytochrome c oxidase Soret band shift signal develops have been monitored. Retardation the ATP-dependent cytochrome c oxidase Soret band shift signal suggests that the probe readily permeates the mitochondrial membrane. The first-order rate law that the glutamate-dependent signal obeys suggests that the rate-limiting step in the development of this signal is the dissociation of the dye from the mitochondrial membrane or the permeation of this membrane by the probe. The faster phase of the ATP-induced signal likely reflects the initial transfer of dye from the bulk aqueous phase followed by a slower probe permeation process that obeys a first-order rate law. This probe appears to distribute across the mitochondrial membrane in accordance with the transmembrane potential as judged by its effect on the ATP-dependent cytochrome c oxidase Soret band shift signal. DiS-C3-(5) also appears to inhibit the NADH dehydrogenase.     

Potentiometric studies on yeast complex III

Potentiometric measurements have been performed on Complex III from bakers' yeast. The midpoint potentials for the b and c cytochromes were measured using room-temperature MCD and liquid-helium temperature EPR. A value of 270 mV was obtained for cytochrome c1, regardless of temperature, while the midpoint potentials found for the two species of cytochrome b varied with temperatures, viz., 62 and -20 mV at room temperature (MCD) compared to 116 and -4 mV at about 10 K (EPR). The midpoint potential of the iron-sulfur center obtained by low-temperature EPR was 286 mV. An abrupt conformational change occurred immediately after this center was fully reduced resulting in a change in EPR line shape. The potentials of the two half-reactions of ubiquinone were measured by following the semiquinone radical signal at 110 K and 23 degrees C. Potentials of 176 and 51 mV were found at low temperature, while values of 200 and 110 mV were observed at room temperature. The midpoint potential of cytochrome c1 was found to be pH independent. The potentials of cytochrome b were also independent of pH when titrations were performed in deoxycholate buffers, while a variation of -30 mV per pH unit was observed for both cytochrome c species in taurocholate buffers. These two detergents also produced different MCD contributions of the two b cytochromes. A decrease in Em of greater than 300 mV was found in potentiometric measurements of cytochrome c1 at high ratios of dye to Complex III. Antimycin does not affect the redox potentials of cytochrome c1 but appears to induce a transition of the low-potential b heme to a high-potential species. This transition is mediated by ubiquinone.     

The tryptophan residues of aspartate transcarbamylase: site-directed mutagenesis and time-resolved fluorescence spectroscopy.

Aspartate transcarbamylase (EC 2.1.3.2) contains two tryptophan residues in position 209 and 284 of the catalytic chains (c) and no such chromophore in the regulatory chains (r). Thus, as a dodecamer [(c3)2(r2)3] the native enzyme molecule contains 12 tryptophan residues. The present study of the regulatory conformational changes in this enzyme is based on the fluorescence properties of these intrinsic probes. Site-directed mutagenesis was used in order to differentiate the respective contributions of the two tryptophans to the fluorescence properties of the enzyme and to identify the mobility of their environment in the course of the different regulatory processes. Each of these tryptophan residues gives two independent fluorescence decays, suggesting that the catalytic subunit exists in two slightly different conformational states. The binding of the substrate analog N-phosphonacetyl-L-aspartate promotes the same fluorescence signal whether or not the catalytic subunits are associated with the regulatory subunits, suggesting that the substrate-induced conformational change of the catalytic subunit is the essential trigger for the quaternary structure transition involved in cooperativity. The binding of the substrate analog affects mostly the environment of tryptophan 284, while the binding of the activator ATP affects mostly the environment of tryptophan 209, confirming that this activator acts through a mechanism different from that involved in homotropic cooperativity.     

The effects of copper depletion on structural aspects of cytochrome c oxidase

The removal of copper from beef heart cytochrome c oxidase by either dialysis against potassium cyanide or by treatment with bathocuproine sulfonate produced changes in the enzyme which are indicative of a spin state transition. In the Soret region of the CD spectrum copper depletion of the enzyme caused a significant decrease in amplitude in combination with a red shift of the peak maximum for oxidized samples, while reduced copper-depleted samples exhibited decreased amplitude and a blue shift of the peak maximum. In the magnetic CD spectra of oxidized copper-depleted samples the peak at 420 nm was shifted to lower wave-length along with a significant increase in amplitude. In reduced samples the peak at 446 nm exhibited a slight red shift concomitant with a substantial decrease in amplitude. The conformational changes indicated by the CD and magnetic CD spectra when copper is removed from the enzyme were supported by the EPR spectra of the NO complex of the reduced copper-depleted enzyme. The removal of copper from cytochrome c oxidase caused the NO complex to exhibit a 3-line splitting pattern of gz in the EPR spectrum instead of the 9 lines seen in the NO complex of the native enzyme. When [15N]NO was used, a 2-line pattern was seen at gz when copper was removed from the enzyme. The changes in the CD and magnetic CD spectra and in the EPR spectra of the NO derivatives of cytochrome c oxidase can be explained by the rearrangement of the axial ligands to iron in cytochrome a3 as a result of copper depletion. These results emphasize the close structural interdependence of the metallic components of this enzyme.     

Presteady-state and steady-state kinetic properties of human cytochrome c oxidase. Identification of rate-limiting steps in mammalian cytochrome c oxidase.

Human cytochrome c oxidase was purified in a fully active form from heart and skeletal muscle. The enzyme was selectively solubilised with octylglucoside and KCl from submitochondrial particles followed by ammonium sulphate fractionation. The presteady-state and steady-state kinetic properties of the human cytochrome c oxidase preparations with either human cytochrome c or horse cytochrome c were studied spectrophotometrically and compared with those of bovine heart cytochrome c oxidase. The interaction between human cytochrome c and human cytochrome c oxidase proved to be highly specific. It is proposed that for efficient electron transfer to occur, a conformational change in the complex is required, thereby shifting the initially unfavourable redox equilibrium. The very slow presteady-state reaction between human cytochrome c oxidase and horse cytochrome c suggests that, in this case, the conformational change does not occur. The proposed model was also used to explain the steady-state kinetic parameters under various conditions. At high ionic strength (I = 200 mM, pH 7.4), the kcat was highly dependent on the type of oxidase and it is proposed that the internal electron transfer is the rate-limiting step. The kcat value of the 'high-affinity' phase, observed at low ionic strength (I = 18 mM, pH 7.4), was determined by the cytochrome c/cytochrome c oxidase combination applied, whereas the Km was highly dependent only on the type of cytochrome c used. Our results suggest that, depending on the cytochrome c/cytochrome c oxidase combination, either the dissociation of ferricytochrome c or the internal electron transfer is the rate-limiting step in the 'high-affinity' phase at low ionic strength. The 'low-affinity' kcat value was not only determined by the type of oxidase used, but also by the type of cytochrome c. It is proposed that the internal electron-transfer rate of the 'low-affinity' reaction is enhanced by the binding of a second molecule of cytochrome c.     

Redox titration of all electron carriers of cytochrome c oxidase by Fourier transform infrared spectroscopy

Electrochemical redox titrations of cytochrome c oxidase from Paraccocus denitrificans were performed by attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy. The majority of the differential infrared absorption features may be divided into four groups, which correlate with the redox transitions of the four redox centers of the enzyme. Infrared spectroscopy has the advantage of allowing one to measure independent alterations in redox centers, which are not well separated, or even observed, by other spectroscopic techniques. We found 12 infrared bands that titrated with the highest observed midpoint redox potential (E(m) = 412 mV at pH 6.5) and which had a pH dependence of 52 mV per pH unit in the alkaline region. These bands were assigned to be linked to the Cu(B) center. We assigned bands to the Cu(A) center that showed a pH-independent E(m) of 250 mV. Two other groups of infrared differential bands reflected redox transitions of the two heme groups and showed a more complex behavior. Each of them included two parts, corresponding to high- and low-potential redox transitions. For the bands representing heme a, the ratio of high- to low-potential components was ca. 3:2; for heme a(3) this ratio was ca. 2:3. Taking into account the redox interactions between the hemes, these ratios yielded a difference in E(m) of 9 mV between the hemes (359 mV for heme a; 350 mV for heme a(3) at pH 8.0). The extent of the redox interaction between the hemes (-115 mV at pH 8.0) was found to be pH-dependent. The pH dependence of the E(m) values for the two hemes was the same and about two times smaller than the theoretical one, suggesting that an acid/base group binds a proton upon reduction of either heme. The applied approach allowed assignment of infrared bands in each of the four groups to vibrations of the hemes, ligands of the redox centers, amino acid residues, and/or protein backbone. For example, the well-known band shift at 1737/1746 cm(-)(1) corresponding to the protonated glutamic acid E278 correlated with oxidoreduction of heme a.