High vectorial proton stoichiometry by cytochrome c oxidase from the thermophilic bacterium PS3 reconstituted in liposomes

The stoichiometry of vectorial H+ ejection, coupled to ferrocytochrome c oxidation by a three-subunit bacterial cytochrome c oxidase (EC 1.9.3.1) from the thermophilic bacterium PS3, was measured. Three methods of measuring the H+/e- ratio were applied to proteoliposomes containing a relatively small amount of PS3 cytochrome oxidase, which showed a relatively low oxidation rate and a very low H+ leakage, as follows: (a) simultaneous measurements of H+ ejection and cytochrome c oxidation upon addition of a yeast ferrocytochrome c pulse, which enable us to calculate the H+/e- ratio as H+ ejected per cytochrome c oxidized; (b) computer simulations to find out the fit for the pH meter trace by changing the H+/e- ratio and the velocity constant of leakage; and (c) two successive measurements of initial rates of H+ movement in the absence and presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone. The H+/e- ratios obtained were 1.39, the 10-s value after ferrocytochrome c addition in (a), 1.35 in (b), and 1.33 in (c). This high H+/e- stoichiometry observed, exceeding 1 and as high as 1.4, is discussed with respect to the controversy of the H+/e- ratio at the cytochrome oxidase site.     

Effects of detergents and cytochrome c binding on scalar and vectorial proton ejection by proteoliposomes containing cytochrome oxidase.

The detergent lauryl maltoside abolishes respiratory control and proton ejection by cytochrome c oxidase-containing proteoliposomes over a narrow concentration range. Expression of cryptic activity (inward-facing oxidase) is released over the same concentration range. Catalytic functions (Vmax. and Km) of the enzyme are not changed by the detergent. Lipid micelles containing detergent bind approximately the same amount of cytochrome c as do vesicles containing an equivalent amount of lipid. Uncoupler-insensitive proton release is seen when proteoliposomes are pulsed with ferrocytochrome c at low ionic strength. Such uncoupler-insensitive acidification is not seen at higher ionic strength, nor with oxygen pulses of anaerobic solutions previously incubated with cytochrome c. Vesicles at low ionic strength catalyse cytochrome c autoxidation; this process can mimic proton re-equilibration in systems that have pumped protons from inside to the bulk phase. Proton re-equilibration following a pulse of cytochrome c or oxygen is multiphasic. The slowest phases are attributed to vesicle heterogeneity, some internal alkali being retained within vesicles of low intrinsic proton permeability. This can be overcome by the addition of either very low levels of carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone or high levels of valinomycin.     

Orientation and reactivity of cytochrome aa3 heme groups in proteoliposomes

Reduction of cytochrome aa₃ in proteoliposomes with ascorbate plus cytochrome c confirms that not more than 55% of the molecules are externally accessible and that the remainder are reduced only on the addition of membrane-permeable N,N,N′,N′tetramethyl-p-henylenediamine. Reduction in the presence of terminal inhibitors such as cyanide, azide, and carbon monoxide shows that likewise 50% of the cytochrome a is accessible and 50% inaccessible. Dithionite reduces part of the cytochrome a₃ in the presence of azide, and none in the presence of cyanide. Methyl viologen, which is somewhat membrane permeable, can reduce part of the cyanide-complexed cytochrome a₃ at low concentrations and all of it at high concentrations. Cytochrome a₃ is therefore also distributed randomly inside and outside the vesicles. Cytochrome c oxidase with externally facing cytochrome a is stimulated to high activity by its membrane association. Its turnover is dependent on the external pH and it is inhibited by external azide; trapping of azide cannot be used to demonstrate the orientation of the cytochrome a₃ hemes associated with externally facing cytochrome a. Cytochrome c oxidase with internally facing cytochrome a is rather sluggishly reactive. Its low activity accounts for the apparent failure of detergents to release extra activity on lysing proteoliposomes. Double reciprocal plots of the reaction of added cytochrome c with proteoliposomes indicate apparent biphasic binding in the energized state, which is abolished upon the addition of uncouplers and valinomycin. But no transmembraneous effect upon the oxidase reaction other than energization has been identified.     

Rates of cyanide binding to the catalytic intermediates of mammalian cytochrome c oxidase, and the effects of cytochrome c and poly(L-lysine).

Rate constants of cyanide binding to 'fast' oxidase have been measured in the fully-oxidised (O), peroxy (P) and ferryl (F) states at pH 8.0. Values of 2.2, 8 and 10 M-1 s-1, respectively, were obtained. Thus, none of these states appears to exhibit a rate that would identify it as the species responsible for the extremely rapid cyanide binding observed during turnover. On the other hand, with 'oxidised' enzyme as prepared, containing a very small fraction of one-electron-reduced (E state) oxidase, a corresponding fraction of enzyme exhibited spectral changes consistent with cyanide binding with a rate constant in excess of 10(4) M-1 s-1. Evidence is presented suggesting that mediation of electron transfer from one-electron-reduced, cyanide-liganded enzyme to free, ferric oxidase, rather than a global protein conformational change of the enzyme, is responsible for the greatly enhanced cyanide binding rates seen in the presence of cytochrome c or poly(L-lysine). Inter-oxidase electron exchange in 'oxidised' enzyme can result in a complicated dependence of the binding rate on cyanide concentration. We have demonstrated that this may give rise to a saturation of the rate of cyanide binding.     

The possible role of the closed-open transition in proton pumping by cytochrome c oxidase: the pH dependence of cyanide inhibition

The rate of oxidation of reduced cytochrome c catalyzed by cytochrome oxidase in the presence and absence of cyanide has been measured spectrophotometrically at pH 5.5, 6.4, 7.4 and 8.3. At the cytochrome c concentration used (272 microM), the uninhibited rate is maximal at pH 6.4 and drops to a value about one sixth of this maximum at pH 8.3. In the presence of cyanide, the rate initially drops rapidly, but with the cyanide concentration used (5.5 microM) there is still a measurable rate of oxidation when maximal inhibition has been reached. This inhibited rate decreases as the pH increases, whereas the apparent rate constant for cyanide binding is almost independent of pH. The results have been analyzed on the basis of a model in which two-electron reduction of the oxidized enzyme triggers a transition from a closed to an open conformation. It is assumed that cyanide can only bind to the open conformation and, furthermore, that rapid internal electron transfer to the dioxygen-reducing site occurs in this state alone. The analysis shows that the true constant for cyanide binding decreases with decreasing pH to a constant value at low pH. It also indicates that the increase in the catalytic constant with decreasing pH is associated with an increase in the rate of the closed-open conformational transition on protonation of the enzyme, and it is proposed that this transition is operative in electron gating in the proton-pump function of the enzyme.     

Reaction of oxidized cytochrome oxidase with cyanide. Effects of pH, cytochrome c and membrane environment

Binding of HCN with ferric beef heart cytochrome oxidase has been studied in submitochondrial particles, as with the enzyme solubilized in detergent or reconstituted into proteoliposomes. Under all conditions, the reaction proceeds via an intermediate and its kinetics can be described by formal parameters Km and kmax in keeping with the Michaelis-type equation. Km of the reaction strongly depends on the enzyme environment; thus it increases 100-1000 fold upon solubilization of cytochrome oxidase but can be subsequently decreased by incorporation of the enzyme in liposomes and by addition of cytochrome c. pH-dependence of the reaction rate shows that, in submitochondrial particles and proteoliposomes as well as in the case of solubilized enzyme supplement with cytochrome c, HCN specifically binds the form of cytochrome oxidase in which a heme-linked ionizable group with pKa 6,5-6,9 is protonated.     

Protonmotive functions of cytochrome c oxidase in reconstituted vesicles. Influence of turnover rate on 'proton translocation'.

1. Oxidation of ferrocytochrome c by cytochrome c oxidase incorporated into proteoliposomes induces a transient acidification of the external medium. This change is dependent on the presence of valinomycin and can be abolished by carbonyl cyanide p-trifluoromethoxyphenylhydrazone or by nigericin. The H+/e- ratio for the initial acidification varies with the internal buffering capacity of the vesicles, and under suitable conditions approaches + 1, the pulse slowly decaying to give a net alkalinity change with H+/e- value approaching -1. 2. Inhibition of cytochrome c oxidase turnover by ferricytochrome c or by azide addition results in ferrocytochrome c-dependent H+ pulses with decreasing H+/e- ratios. The rate of the initial H+ production remains higher than the rate of equilibration of the pH gradient, indicating an intrinsic dependence of the H+/e- ratio on enzyme turnover. The final net alkalinity changes are relatively unaffected by turnover inhibition.     

Measurements of the proton motive force generated by cytochrome c oxidase from Bacillus subtilis in proteoliposomes and membrane vesicles

Cytochrome c oxidase from Bacillus subtilis was reconstituted in liposomes and its energy-transducing properties were studied. The reconstitution procedure used included Ca2+-induced fusion of pre-formed membranes. The orientation of the enzyme in liposomes is influenced by the phospholipid composition of the membrane. Negatively charged phospholipids are essential for high oxidase activity and respiratory control. Analyses of the proteoliposomes by gel filtration, density gradient centrifugation and electron microscopy indicated a heterogeneity of the proteoliposomes with respect to size and respiratory control. Cytochrome c oxidase activity in the proteoliposomes resulted in the generation of a proton motive force, internally negative and alkaline. In the presence of the electron donor, ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine/cytochrome c or ascorbate/phenazine methosulphate, the reconstituted enzyme generated an electrical potential of 84 mV which was increased by the addition of nigericin to 95 mV and a pH gradient of 32 mV which was increased by the addition of valinomycin to 39 mV. Similar results were obtained with beef-heart cytochrome c oxidase reconstituted in liposomes. The maximal proton motive force which could be generated, assuming no endogenous ion leakage, varied over 110-140 mV. From this the efficiency of energy transduction by cytochrome c oxidase was calculated to be 18-23%, indicating that the oxidase is an efficient proton-motive-force-generating system.     

The reactions of Pseudomonas cytochrome c-551 oxidase with potassium cyanide.

The binding of cyanide to both oxidized and ascorbate-reduced forms of Pseudomonas cytochrome c-551 oxidase was investigated. Spectral studies on the oxidized enzyme and its apoprotein showed that the ligand can bind to both the c and d, haem components of the molecule, and kinetic observations indicated that both chromophores reacted, under a variety of conditions, with very similar rates. Cyanide combination velocities were dependent on ligand concentration, and increasing the pH also accelerated the reaction; the second-order rate constant was estimated as approx. 0.2M-1 . s-1 at pH 7.0. The binding of cyanide to the protein was observed to have a considerable influence on reduction of the enzyme by ascorbate. Spectral and kinetic observations have revealed that the species haem d13+-cyanide and any unbound haem c may react relatively rapidly with the reductant, but the behaviour of cyanide-bound haem c indicates that it may not be reduced without prior dissociation of the ligand, which occurs relatively slowly. The reaction of reduced Pseudomonas cytochrome oxidase with cyanide is radically different from that of the oxidized protein. In this case the ligand only binds to the haem d1 component and reacts much more rapidly. Stopped-flow kinetic measurements showed the binding to be biphasic in form. Both the rates of these processes were dependent on cyanide concentration, with the fast phase having a second-order rate constant of 9.3 X 10(5) M-1 . s-1 and the slow phase one of 2.3 X 10(5) M-1 . s-1. The relative proportions of the two phases also showed a dependency on cyanide concentration, the slower phase increasing as the cyanide concentration decreased. Computer simulations indicate that a reaction scheme originally proposed for the reaction of the enzyme with CO is capable of providing a reasonable explanation of the experimental results. Static-titration data of the reduced enzyme with with cyanide indicated that the binding was non-stoicheiometric, the ligand-binding curve being sigmoidal in shape. A Hill plot of the results yielded a Hill coefficient of 2.6.     

Cyanide stimulated dissociation of chloride from the catalytic center of oxidized cytochrome c oxidase

A comparison of bovine cytochrome c oxidase isolated in the presence and the absence of chloride salts reveals that only enzyme isolated in the presence of chloride salts is a mixture of a complex of oxidized enzyme with chloride (CcO.Cl) and chloride-free enzyme (CcO). Using a spectrophotometric method for chloride determination, it was shown that CcO.Cl contains one chloride ion that is released into the medium by a single turnover or by cyanide binding. Chloride is bound slowly within the heme a(3)-Cu(B) binuclear center of oxidized enzyme in a manner similar to the binding of azide. The pH dependence of the dissociation constant for the formation of the CcO.Cl complex reveals that chloride binding proceeds with the uptake of one proton. With both forms of the enzyme the dependence of the rate of reaction for cyanide binding upon cyanide concentration asymptotes a limiting value indicating the existence of an intermediate. With CcO.Cl this limiting rate is 10(3) higher than the rate of the spontaneous dissociation of chloride from the binuclear center and we propose that the initial step is the coordination of cyanide to Cu(B) and in this intermediate state the rate of dissociation of chloride is substantially enhanced.     

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.     

Formation of pH and potential gradients by the reconstituted Azotobacter vinelandii cytochrome bd respiratory protection oxidase.

To directly characterize the bioenergetic properties of the cytochrome bd terminating branch of the Azotobacter vinelandii electron transport chain, the purified cytochrome bd oxidase was reconstituted into a phospholipid environment consisting of phosphatidylethanolamine and phosphatidylglycerol (3:1). The average diameter of the proteoliposomes after extrusion through a polycarbonate membrane was 94 +/- 4 nm. Initiation of respiration upon the addition of 20 microM ubiquinone-1 to proteoliposomes loaded with the pH-sensitive dye pyranine resulted in an immediate alkalization of the vesicle lumen by an average pH change of 0.11 unit. This pH gradient was readily collapsed upon the addition of nigericin, carbonyl cyanide p-(tri-fluoromethoxy) phenyl-hydrazone, gramicidin, Triton X-100, or 2-heptyl-4-hydroxyquinoline N-oxide (HQNO). Proteoliposomal respiration initiated in the presence of the potentiometric membrane dye rhodamine 123 caused the generation of a transmembrane potential; the potential was collapsed upon the addition of either valinomycin or HQNO. The formation of both pH and potential gradients during turnover demonstrates that the A. vinelandii cytochrome bd oxidase is coupled to energy conservation in vivo.     

Palmitate decreases proton pumping of liver-type cytochrome c oxidase.

The H+/e- stoichiometry of reconstituted cytochrome c oxidase from bovine kidney, containing subunit VIaL (liver type), is 0.5 under standard conditions but 1.0 on addition of 1% cardiolipin to the lipid mixture (asolectin). Low concentrations of palmitate (half-maximal effect at 0.5 microm), but not laurate, myristate, stearate, oleate, 1-hexadecanol, palmitoyl glycerol and palmitoyl CoA, decreased the H+/e- ratio in the presence of cardiolipin from 1.0 to 0.5, accompanied by an increase of coupled, but not of uncoupled respiration of proteoliposomes. Cardiolipin and palmitate did not influence the H+/e- stoichiometry and respiration of reconstituted cytochrome c oxidase from bovine heart, containing subunit VIaH (heart-type). The H+/e- stoichiometry of the heart enzyme, however, is decreased from 1.0 to 0.5 by 5 mm intraliposomal ATP (instead of 5 mm ADP). It is assumed that palmitate binds to subunit VIaL. The partial uncoupling of proton pumping in cytochrome c oxidase is suggested to participate in mammalian thermogenesis.     

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.     

Solvent proton relaxation studies of cytochrome c oxidase solutions

The interaction of solvent water protons with the bound paramagnetic metal ions of beef heart cytochrome c oxidase has been examined. The observed proton relaxation rates of enzyme solutions had a negative temperature dependence, indicating a rapid exchange between solvent protons in the coordination sphere of the metal ions and bulk solvent. An analysis of the dependence of the proton relaxation rate on the observation frequency indicated that the correlation time, which modulates the interaction between solvent protons and the unpaired electrons on the metal ions, is due to the electron spin relaxation time of the heme irons of cytochrome c oxidase. This means that at least one of the hemes is exposed to solvent. The proton relaxation rate of the oxidized enzyme was found to be sensitive to changes in ionic strength and to changes in the spin states of the metal ions. Heme a3 was found to be relatively inaccessible to bulk solvent. Partial reduction of the enzyme caused a slight increase in the relaxation rate, which may be due to a change in the antiferromagnetic coupling between two of the bound paramagnetic centers. Further reduction resulted in a decreased relaxation rate, and the fully reduced enzyme was no longer sensitive to changes in ionic strength. The binding of cytochrome c to cytochrome c oxidase had little effect on the proton relaxation rates of oxidized cytochrome oxidase indicating that cytochrome c binding has little effect on solvent accessibility to the metal ion sites.     

Effect of heat treatment on oxidase activity and proton-pumping capability of proteoliposome-incorporated beef heart cytochrome aa3

By incubating beef heart cytochrome c oxidase at 43-45 degrees C, selective inactivation of the H+-pumping function is possible without affecting cytochrome c oxidase activity; proteoliposomes reconstituted with heated enzyme (43.5 degrees C for 60 min at pH 7.0) showed an apparent H+/e- ratio of only 0.3 and a turnover with cytochrome c plus ferrocyanide as substrate of 20 s-1, while those with the intact enzyme showed an apparent H+/e- ratio somewhat greater than 1.0 and a turnover of 19 s-1. This decrease in the H+/e- ratio could not be attributed to a stimulation of H+ permeability upon heating, since the respiratory control ratio and the magnitude of membrane potential formation remained almost the same in the two cases. A pH-dependent Em (midpoint redox potential) change of cytochrome a in the presence of cyanide was still observed after the heat treatment. Heating induced a small spectral shift in the Soret region of the oxidized (resting) enzyme; the peak of the heated enzyme was at 421 nm, while that of the intact enzyme was at 419 nm. The spectral shift obtained by pulsing the enzyme with oxygen under turnover conditions is also altered.     

A re-examination of the reactions of cyanide with cytochrome c oxidase

Experiments were performed to examine the cyanide-binding properties of resting and pulsed cytochrome c oxidase in both their stable and transient turnover states. Inhibition of the oxidation of ferrocytochrome c was monitored as a function of cyanide concentration. Cyanide binding to partially reduced forms produced by mixing cytochrome c oxidase with sodium dithionite was also examined. A model is presented that accounts fully for cyanide inhibition of the enzyme, the essential feature of which is the rapid, tight, binding of cyanide to transient, partially reduced, forms of the enzyme populated during turnover. Computer fitting of the experimentally obtained data to the kinetic predictions given by this model indicate that the cyanide-sensitive form of the enzyme binds the ligand with combination constants in excess of 10(6) M-1 X s-1 and with KD values of 50 nM or less. Kinetic difference spectra indicate that cyanide binds to oxidized cytochrome a33+ and that this occurs rapidly only when cytochrome a and CuA are reduced.     

Heat treatment of cytochrome c oxidase perturbs the CuA site and affects proton pumping behavior

It has been previously reported that mild heat treatment (43 degrees C for ca. 60 min) abolishes the proton pumping activity of cytochrome c oxidase while leaving the oxidase activity and cytochromes a and a3 unperturbed [Sone, N., & Nicholls, P. (1984) Biochemistry 23, 6550-6554]. We herein describe the effects of this heat treatment on the electron paramagnetic resonance (EPR) and optical absorption signatures of the redox-active metal centers in the enzyme. We find that heat treatment of the oxidized enzyme causes a local structural perturbation at the CuA site. After heat treatment, the enzyme sample contains three subpopulations, each of which has a different structure at CuA. These include (i) native CuA, (ii) a type 2 copper species similar to the one produced by chemical modification by p-(hydroxymercuri)benzoate (pHMB) [Gelles, J., & Chan, S. I. (1985) Biochemistry 24, 3963-3972], and (iii) a novel type 1 copper species. In addition to changes at the CuA site, we find that heat treatment results in accelerated cyanide binding and the removal of subunit III. If the cytochrome c oxidase is heat treated while fully reduced, none of these changes are observed except for subunit III depletion. Furthermore, partial (CO mixed-valence derivative) reduction of the enzyme as well as ligand binding to cytochrome a3 also protects the enzyme against the heat-induced changes, indicating that the oxygen binding site plays a role in stabilizing the CuA site against structural perturbations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of reduction of heme a and Cu(A) on the oxidized catalytic center of cytochrome c oxidase: insight from organic solvents

Purified bovine heart cytochrome c oxidase (CcO) has been extracted from aqueous solution into hexane in the presence of phospholipids and calcium ions. In extracts, CcO is in the so-called "slow" form and probably situated in reverse micelles. At low water:phospholipid molar ratios, electron transfer from reduced heme a and Cu(A) to the catalytic center is inhibited and both heme a3 and Cu(B) remain in the oxidized state. The rate of binding of cyanide to heme a3 in this oxidized catalytic center is, however, dependent on the redox state of heme a and Cu(A). When heme a and Cu(A) are reduced, the rate is increased 20-fold compared to the rate when these two centers are oxidized. The enhanced rate of binding of cyanide to heme a3 is explained by the destabilization of an intrinsic ligand, located at the catalytic site, that is triggered by the reduction of heme a and Cu(A).     

Kinetic studies on the binding of cyanide to oxygenated cytochrome c oxidase.

The reaction of cyanide with oxygenated cytochrome c oxidase was followed by means of flow-flash techniques. The oxygenated form, produced after photolysis of the partially reduced CO complex in the presence of cyanide and O2, shows cyanide-binding properties distinct from those of both the oxidized and the reduced forms of the protein. The binding is a single process (k = 22M-1-S-1) linearly dependent on cyanide concentration to as high as 75 mM. It is suggested that the oxygenated form is a conformational variant of the oxidized protein.