The invisible copper of cytochrome c oxidase. pH and ATP dependence of its midpoint potential and its role in the oxygen reaction.

Formation of the CO compound has been studied in intact mitochondria, submitochondrial particles and isolated cytochrome oxidase. The reaction requires the prior reduction of both cytochrome a₃ and one other single-electron acceptor. It is inferred that the second acceptor is the “invisible” copper which is undetectable by both optical and spin resonance spectroscopy. The overall process can be viewed as two single electron steps plus a ligand binding reaction. At high concentrations of CO, when titrations are performed at oxidation-reduction potentials significantly above the midpoints of either cytochrome a₃ or “invisible” copper, appearance of the CO compound follows a strict n = 2 (2-electron) relationship. Its midpoint potential is also dependent on the prevailing concentration of CO and is increased by approx. 30 mV for each tenfold increase in the level of CO. At redox potentials approaching the midpoints of cytochrome a₃ or “invisible” copper, significant deviations from n = 2 behavior are apparent which are readily detectable experimentally using low CO concentrations.A mathematical analysis of this model is presented and the oxidation-reduction properties of the CO compound are utilized to determine the midpoint potential of the “invisible” copper. This value is estimated to be 340 ± 10 mV at pH 7.8, independent of pH and the prevailing $\text{sol}\text{[ATP]}\text{[ADP]}\text{× [}\text{P}{}_1\text{]}$ ratio.By analogy with the observations on CO binding, the primary intermediate in the oxidase reaction with oxygen is concluded to be a bridged a₃²⁺-O₂-Cu¹⁺ complex. The initial reduction of molecular oxygen can then proceed via a thermodynamically favorable two-electron step to form a bridged peroxide intermediate. Subsequent reduction to water may later occur by way of two single-electron steps or one two-electron step.     

Reaction of CO with cytochrome c oxidase. Titration of the reaction site with chemical oxidant and reductant

The number of reducing equivalents required to form the reduced cytochrome a₃-CO compound has been determined for suspensions of submitochondrial particles and for isolated cytochrome c oxidase. Anaerobic preparations were titrated reductively with NADH and oxidatively with O₂ in the presence of high concentrations of CO (0.4 to 0.8 mM) while monitoring reduction of cytochrome a and the formation of the reduced cytochrome a₃-CO compound by their characteristic absorbance changes. Analysis of the titration data show that 2.0±0.3 and 2.1±0.2 reducing equivalents per mol of cytochrome oxidase (per cytochrome a) are required for formation of the reduced cytochrome a₃-CO compound in submitochondrial particles and isolated cytochrome c oxidase, respectively. In each case, the formation of the CO compound is proportional to the number of equivalents accepted by the preparation, indicating that the two equivalents are equal and the effective n value for the reaction is 2.0. Potentiometric titrations of cytochrome c oxidase using the cobalt orthophenanthrolene complex (E_{m, 7.0} = 0.37 V) as mediator give the same half-reduction potential values for cytochrome a and a₃ as those obtained using the ferro-ferricyanide couple. The formation of the reduced cytochrome a₃-CO compound at pH 7.0, in the presence of 0.6 mM CO and with CO-orthophenanthrolene as mediator occurs with a half-reduction potential of 0.45 V and requires two electrons. These data confirm and extend the observation of Lindsay et al. (Arch. Biochim. Biophys. (1975) 169, 492–505) that both the “invisible” copper and cytochrome a₃ must be reduced in order for CO to bind with high affinity.     

The use of a maleimido spin label to detect conformational changes in cytochrome c oxidase.

Spin labeling with a maleimido spin label has been used to investigate conformational changes of bovine cytochrome c oxidase. These experiments show that the spin label is immobilized to a lesser degree when the enzyme is in the “oxygenated” form than it is in the oxidized state and support the view that the oxygenated form is a conformational variant. Experiments in which the maleimido spin-labeled cytochrome c oxidase was titrated with H₂O₂ reveal that the peroxide-treated enzyme, although possessing an absorption spectrum similar to that of the oxygenated form, has an electron paramagnetic resonance (epr) spectrum that is different from that of either the oxygenated form or the oxidized state. Extremes of pH cause a marked decrease in the degree of immobilization of maleimido spin labels bound to the oxidase. Alterations in the epr spectrum are reversible if the pH is held between 5.3 and 10.2 but are irreversible outside that range. Urea and guanidine hydrochloride also decrease the immobilization of the spin labels bound to the oxidase. The nature of the epr spectra indicates that under these conditions the enzyme assumes a more open conformation. Exposure to concentrations of sodium dodecyl sulfate as high as 10% does not result in as much loss of the immobilization as with urea or guanidine. Detergents such as cholate, Tween 80, and Triton X-100 have no significant effect on the epr spectrum of maleimido spin-labeled cytochrome c oxidase.     

Modulation of the electron-proton coupling at cytochrome a by the ligation of the oxidized catalytic center in bovine cytochrome c oxidase

Cytochrome a was suggested as the key redox center in the proton pumping process of bovine cytochrome c oxidase (CcO). Recent studies showed that both the structure of heme a and its immediate vicinity are sensitive to the ligation and the redox state of the distant catalytic center composed of iron of cytochrome a₃ (Fe_a3) and copper (Cu_B). Here, the influence of the ligation at the oxidized Fe_a3³⁺–Cu_B²⁺ center on the electron–proton coupling at heme a was examined in the wide pH range (6.5-11). The strength of the coupling was evaluated by the determination of pH dependence of the midpoint potential of heme a (E_m(a)) for the cyanide (the low-spin Fe_a3³⁺) and the formate-ligated CcO (the high-spin Fe_a3³⁺). The measurements were performed under experimental conditions when other three redox centers of CcO are oxidized. Two slightly differing linear pH dependencies of E_m(a) were found for the CN– and the formate–ligated CcO with slopes of −13 mV/pH unit and −23 mV/pH unit, respectively. These linear dependencies indicate only a weak and unspecific electron–proton coupling at cytochrome a in both forms of CcO. The lack of the strong electron–proton coupling at the physiological pH values is also substantiated by the UV–Vis absorption and electron–paramagnetic resonance spectroscopy investigations of the cyanide–ligated oxidized CcO. It is shown that the ligand exchange at Fe_a³⁺ between His–Fe_a³⁺–His and His–Fe_a³⁺–OH⁻ occurs only at pH above 9.5 with the estimated pK >11.0.     

An EPR signal from the "invisible" copper of cytochrome oxidase.

Sulfide is both an inhibitor and a slow reductant of oxidized cytochrome $\text{c}$ oxidase. When the enzyme is exposed to sulfide for short times (one minute or less) and frozen, the resultant electron paramagnetic resonance (EPR) signals show clearly: low spin heme a, low spin heme a₃, the usual “EPR detectable” Cu²⁺ signal (g_⊥ = 2.17, g_⊥ = 2.03), and a new Cu²⁺ signal superimposed on the same region, with (g_⊥ ～ 2.19, g_⊥ = 2.05). This new signal presumably arises because the antiferromagnetic coupling postulated to exist between the iron atom of heme a₃ and this copper is disrupted when heme a₃ is driven to a low spin state by sulfide. The implications of this result with respect to models of the O₂-binding site and redox geometry of oxidase are briefly discussed.     

Heme-copper and heme-heme interactions in the cytochrome bo-containing quinol oxidase of Escherichia coli

The cytochrome bo quinol oxidase of Escherichia coli is one of two respiratory O2 reductases which the bacterium synthesizes. The enzyme complex contains copper and 2 mol of b-type heme. Electron paramagnetic resonance (epr) spectroscopy of membranes from a strain having amplified levels of this enzyme complex reveals signals from low- and high-spin b-type hemes, but the copper, now established as a component of the oxidase, is not directly detectable by epr. The high-spin signal from the cytochrome bo complex, which we attribute to cytochrome o, when titrated potentiometrically, gives a bell-shaped curve. The low potential side of this curve is biphasic (Em7 approximately 180 and 280 mV) and corresponds to the reduction/oxidation of the cytochrome(s). The high potential side of the bell-shaped curve is monophasic (Em7 approximately 370 mV) and is proposed to be due to reduction/oxidation of a copper center which, when in the Cu(II) form, is tightly spin-coupled to a heme, probably cytochrome o, resulting in a net even spin system and loss of the epr spectrum. The low-spin cytochrome b titrates biphasically with Em7 values of approximately 180 and 280 mV, similar to the high-spin component but without the loss of signal at high potentials.     

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.     

Heme-heme interaction in cytochrome c oxidase: the cooperativity of the hemes of cytochrome c oxidase as evidenced in the reaction with CO

Isolated and purified cytochrome c oxidase from beef heart muscle mitochondria (Kuboyama et al. (1972) J. Biol. Chem.247, 6375–6383) is shown to be very similar to the hemoprotein in situ with respect to its EPR absorption properties and the half-reduction potentials of the hemes and copper. The half-reduction potentials of cytochromes a and a₃ in the purified cytochrome c oxidase are 205 mV and 360 mV, respectively, and these values are the same in the presence and absence of cytochrome c.Low-temperature EPR spectra show that the binding of CO to reduced cytochrome a₃ changes the oxidized cytochrome a from high spin (g 6) to low spin (g 3). In samples at 5–8 °K the photodissociation of the reduced cytochrome a₃CO compound shifts the spectrum of the oxidized low-spin cytochrome a to a lower g value and converts approximately 5% of the low-spin form to a high-spin form. The heme-heme interaction demonstrated in this reaction is very fast as evidenced by the fact that even at 5 °K the measured change in oxidized cytochrome is complete within 5 msec.     

Studies on the orientations of the mitochondrial redox carriers I. Orientation of the hemes of cytochrome c oxidase with respect to the plane of a cytochrome oxidase-lipid model membrane

The liganded derivatives of mitochondrial cytochrome c oxidase have been prepared in hydrated oriented multilayers of membranous cytochrome c oxidase. The optical spectra of the liganded derivatives recorded at an angle of 45° between the incident light beam and the normal to the planes of the membranes in the multilayers show dichroic ratios of almost 2 in the visible region and 1.2–1.4 in the Soret region. The dichroic ratios were found to be similar for both cytochromes a and a₃. Electron paramagnetic resonance spectra of the azide, sulfide, and formate complexes of cytochrome c oxidase obtained as a function of the orientation of the applied magnetic field relative to the planes of the membranes in the multilayer confirm the optical data and demonstrate that both hemes of cytochrome c oxidase are oriented such that the angle between the heme normal and the membrane normal is approximately 90°.     

Electron-transfer processes in carboxy-cytochrome c oxidase after photodissociation of cytochrome a2+3 · CO

Under continuous illumination the CO binding curve of reduced carboxy-cytochrome c oxidase maintains the shape of the binding curve in the dark. The apparent dissociation constant calculated from the binding curves at various light intensities is a linear function of the light intensity.Marked differences are observed between the light-induced difference spectra of the fully reduced carboxy-cytochrome c oxidase and the mixed-valence carboxy-cytochrome c oxidase. These differences are enhanced in the presence of ferricyanide as an electron acceptor and are explained by partial oxidation of cytochrome a₃ in the mixed-valence enzyme after photodissociation.Upon addition of CO to partially reduced formate cytochrome c oxidase (a²⁺a³⁺₃ · HCOOH) the cytochrome a²⁺₃ · CO compound is formed completely with a concomitant oxidation of cytochrome a and the Cu associated with cytochrome a. During photodissociation of the CO compound the formate rebinds to cytochrome a₃ and cytochrome a and its associated Cu are simultaneously reduced. These electron transfer processes are fully reversible since in the dark the a³⁺₃ · HCOOH compound is dissociated slowly with a concomitant formation of the a²⁺₃ · CO compound and oxidation of cytochrome a.When these experiments are carried out in the presence of cytochrome c, both cytochrome c and cytochrome a are reduced upon illumination of the mixed-valence carboxy-cytochrome c oxidase. In the dark both cytochrome c and cytochrome a are reoxidized when formate dissociates from cytochrome a₃ and the a²⁺₃ · CO compound is formed back. Thus, in this system we are able to reverse and to modulate the redox state of the different components of the final part of the respiratory chain by light.     

Biochemical and biophysical studies on cytochrome c oxidase XVI. Circular dichroic study of cytochrome c oxidase and its ligand complexes

1. CD spectra of cytochrome c oxidase have been determined both in the absence and presence of the extrinsic ligands CO, NO, cyanide and azide.2. CO and NO affect the CD spectrum of cytochrome c oxidase in a similar way.3. Cyanide and azide also affect the CD spectrum of cytochrome c oxidase in a similar way, but distinctly different from CO and NO.4. From the CD spectra of the oxidized and reduced enzyme, in the presence and absence of extrinsic ligands, CD difference spectra (reduced minus oxidized) are calculated for the so-called cytochrome a and cytochrome a₃ moieties of the enzyme.5. These spectra are largely dependent on the extrinsic ligand used. It is therefore concluded that these spectra do not represent independent cytochrome a and cytochrome a₃ difference spectra, but that heme-heme interactions occur within the cytochrome c oxidase molecule, in such a way that binding of a ligand to one of the heme a groups of cytochrome c oxidase affects the spectral properties of the other heme a group.6. As a consequence, ligand-binding studies cannot give information as to the pre-existence of separate cytochrome a and cytochrome a₃ moieties in the absence of extrinsic ligands.     

Differential affinity of BsSCO for Cu(II) and Cu(I) suggests a redox role in copper transfer to the CuA center of cytochrome c oxidase

SCO (synthesis of cytochrome c oxidase) proteins are involved in the assembly of the respiratory chain enzyme cytochrome c oxidase acting to assist in the assembly of the Cu_A center contained within subunit II of the oxidase complex. The Cu_A center receives electrons from the reductive substrate ferrocytochrome c, and passes them on to the cytochrome a center. Cytochrome a feeds electrons to the oxygen reaction site composed of cytochrome a₃ and Cu_B. Cu_A consists of two copper ions positioned within bonding distance and ligated by two histidine side chains, one methionine, a backbone carbonyl and two bridging cysteine residues. The complex structure and redox capacity of Cu_A present a potential assembly challenge. SCO proteins are members of the thioredoxin family which led to the early suggestion of a disulfide exchange function for SCO in Cu_A assembly, whereas the copper binding capacity of the Bacillus subtilis version of SCO (i.e., BsSCO) suggests a direct role for SCO proteins in copper transfer. We have characterized redox and copper exchange properties of apo- and metalated-BsSCO. The release of copper (II) from its complex with BsSCO is best achieved by reducing it to Cu(I). We propose a mechanism involving both disulfide and copper exchange between BsSCO and the apo-Cu_A site. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.     

Oxidation-reduction potentials of respiratory chain components in ThiobacillusA2

(1) Cells of ThiobacillusA₂ grown chemoautotrophically on thiosulfate or heterotrophically on succinate with oxygen contained b-, c-, o-, a- and a₃-type cytochromes. The amount of cytochrome per mg of cell protein was much greater in thiosulfate-grown cells and differences in the relative concentrations of cytochromes were observed for the different growth conditions. (2) The half-reduction potentials at pH 7.0 (E_m,7.0) and spectral maxima of c-, b-, a- and a₃-type cytochromes were similar in cells grown aerobically with thiosulfate or with succinate as the growth substrate. (3) The half-reduction potential of the ‘invisible’, or high-potential copper, as determined from the potentiometric behavior of the carbon monoxide-reduced cytochrome a₃ complex at pH 8.0, was 365 mV. (4) Reducing equivalents from thiosulfate appear to enter the respiratory chain at the cytochrome c level; however, studies in cell-free extracts were limited due to a loss in respiratory activity with thiosulfate as a substrate upon cell disruption.     

Responses of the a3 component of cytochrome c oxidase to substrate and ligand addition

We have previously described a transient high spin ferric heme species in cytochrome c oxidase (EC 1.9.3.1) which represents a³⁺₃ (Beinert, H. and Shaw, R.W. (1977) Biochim. Biophys. Acta 462, 121–130), and can be detected and quantitatively determined by EPR. We have now used our ability to generate this species to study reactions of a³⁺₃ with substrates and ligands and also responses to pH changes. This was accomplished by multiple rapid mixing and freezing techniques in conjunction with low temperature EPR and optical reflectance spectroscopies. The substrates used were O₂ and ferrocytochrome c and the ligands cyanide, sulfide, azide and carbon monoxide. Contrary to the oxidized, resting form of the enzyme, the transient high spin species of a³⁺₃ reacts within <10 ms stoichiometrically with cyanide and sulfide and at a slower rate with azide. The transient a³⁺₃ species responds to O₂ and CO by changes in signal size or shape, although no oxidoreduction is involved, indicating that a³⁺₃ registers the presence of these gases. The high spin signal of the transient species is readily abolished by ferrocytochrome c or on raising the pH. Decreasing the pH induces a shift from the rhombic towards the axial component of the signal. Since the responses to CO and pH are analogous for the rhombic transient species to those observed with the rhombic high spin ferric heme species produced on partial reduction, it is suggested that the rhombic signals represent a³⁺₃ in either case. In all these experiments, in which EPR detectable a³⁺₃ was observed in large yield, no extra signals for copper or correspondingly increased intensity in the copper signal at g = 2 were seen. The relationship is discussed of the obviously reactive transient species of a³⁺₃ to other ‘activated’ species that have been reported and to the oxidized resting form of the enzyme, which is known to react only slowly with ligands and to respond sluggishly to substrate.     

The oxidation-reduction potentials of the hemes and copper of cytochrome oxidase from beef heart

The oxidation-reduction potentials of the heme and copper components of isolated beef heart cytochrome oxidase have been studied by potentiometric techniques. In highly purified preparations the two heme components give a single titration curve with a midpoint potential at pH 7.0 (E_m^7.0) of +285 mV and an n value of 0.5. In partially purified preparations the heme components could be resolved into a high potential cytochrome (a₃) (E_m^7.0 = +375 mV, an n value of 1.0) and a low potential cytochrome (a) (E_m^7.0 = +225 mV, an n value of 1.0). In general, with decrease in enzymatic activity the E_m^7.0 of the high potential component becomes more negative.     

EPR studies of cytochrome aa3 from Sulfolobus acidocaldarius. Evidence for a binuclear center in archaebacterial terminal oxidase.

The purified cytochrome aa3-type oxidase from Sulfolobus acidocaldarius (DSM 639) consists of a single subunit, containing one low-spin and one high-spin A-type hemes and copper [Anemüller, S. and Sch?fer, G. (1990) Eur. J. Biochem. 191, 297-305]. The enzyme metal centers were investigated by electron paramagnetic resonance spectroscopy (EPR), coupled to redox potentiometry. The low-spin heme EPR signal has the following g-values: gz = 3.02, gy = 2.23 and gx = 1.45 and the high-spin heme exhibits an almost axial spectrum (gy = 6.03 and gx = 5.97, E/D < 0.002). In the enzyme as isolated the low-spin resonance corresponds to 95 +/- 10% of the enzyme concentration, while the high-spin signal accounts for only 40 +/- 5%. However, taking into account the redox potential dependence of the high-spin heme signal, this value also rises to 95 +/- 10%. The high-spin heme signal of the Sulfolobus enzyme shows spectral characteristics distinct from those of the Paracoccus denitrificans one: it shows a smaller rhombicity (gy = 6.1 and gx = 5.9, E/D = 0.004 for the P. denitrificans enzyme) and it is easier to saturate, having a half saturation power of 148 mW compared to 360 mW for the P. denitrificans protein, both at 10 K. The EPR spectrum of an extensively dialyzed and active enzyme sample containing only one copper atom/enzyme molecule does not display CuA-like resonances, indicating that this enzyme contains only a CUB-type center. The EPR-redox titration of the high-spin heme signal, which is assigned to cytochrome a3, gives a bell shaped curve, which was simulated by a non-interactive two step redox process, with reduction potentials of 200 +/- 10 mV and 370 +/- 10 mV at pH = 7.4. The decrease of the signal amplitude at high redox potentials is proposed to be due to oxidation of a CUB(I) center, which in the CUB(II) state is tightly spin-coupled to the heme a3 center. The reduction potential of the low-spin resonance was determined using the same model as 305 +/- 10 mV at pH = 7.4 by EPR redox titration. Addition of azide to the enzyme affects only the high-spin heme signal, consistent with the assignment of this resonance to heme a3. The results are discussed in the context of the redox center composition of quinol and cytochrome c oxidases.     

Effect of membrane potential on equilibrium poise between cytochromea and cytochromec in rat liver mitochondria

The object of this work was to test the suggestion that the equilibrium poise between cytochromea and cytochromec in mitochondria might be influenced by the membrane potential.

1. The midpoint potentials of cytochromes (c+c ₁) and cytochromea (CO present) were found to be 250 mV and 245 mV, respectively, by equilibrating rat liver mitochondria with mixtures of ferrocyanide and ferricyanide anaerobically in presence of antimycin A and measuring the redox state of the cytochromes spectrophotometrically. In absence of CO, cytochrome oxidase gave an anomalous redox titration curve with a “midpoint” at about 275 mV.
2. When the mitochondria were equilibrated with ferricyanide/ferrocyanide, the redox poise of cytochromea (CO present) and of cytochromes (a+a ₃) but not of cytochromes (c+c ₁) was dependent on the sign and magnitude of the membrane potential developed by treating the mitochondria as follows: by adding ATP, by chaging the composition of the suspension medium so as to vary the Donnan or Nernst potential, by adding valinomycin in a medium of low K⁺ ion content, or by adding a pulse of acid or alkali when the membrane was made permeable to protons with FCCP.
3. The findings agree with the suggestion that the respiratory chain is arranged across the cristae membrane with cytochromesc ₁ andc in contact with the outer phase and cytochromesa anda ₃ plugged through, so that the equilibrium distribution of electrons between thec anda cytochromes is influenced by the electric field across the membrane.