The interconversion between monomeric and dimeric bovine heart cytochrome c oxidase

Monomers and dimers of bovine heart cytochrome c oxidase (EC 1.9.3.1.) were separated by gel filtration chromatography on Ultrogel AcA 34 or by sucrose gradient centrifugation. Factors influencing the interconversion of the two aggregation states of this enzyme were analyzed. At very low ionic strength, in the presence of dodecyl maltoside, monomers were the main species. Salts appeared to stabilize the dimeric form, divalent cations being more efficient than monovalent. High enzyme concentrations favoured the formation of dimers, also at low ionic strength. The type of detergent had a strong influence on the monomer-dimer interconversion; in Triton X-100 and dodecyl maltoside (at high ionic strength) cytochrome c oxidase was homogenously dispersed in its dimeric form, while in Tween-80 gel filtration showed only large particles eluting in the void volume. In cholate monomers and aggregates were observed but no dimers. The aggregation state had an influence on the steady state kinetics of the ferrocytochrome c oxidase activity. Monomers showed linear Eadie-Hofstee plots, whilst the dimeric and aggregated enzyme gave nonlinear Eadie-Hofstee plots. Ionic strength, enzyme concentration and type of detergent were affecting the enzyme's kinetics in a way consistent with the molecular form obtained by the gel filtration or sedimentation analysis. The data support a negative cooperative mechanism for the interaction of cytochrome c with the dimeric enzyme, as proposed earlier (K.A. Na?ecz et al., (1983) Biochem. Biophys. Res. Commun., 114, 822-828).     

Yeast cytochrome c oxidase: a model system to study mitochondrial forms of the haem-copper oxidase superfamily

The known subunits of yeast mitochondrial cytochrome c oxidase are reviewed. The structures of all eleven of its subunits are explored by building homology models based on the published structures of the homologous bovine subunits and similarities and differences are highlighted, particularly of the core functional subunit I. Yeast genetic techniques to enable introduction of mutations into the three core mitochondrially-encoded subunits are reviewed.     

The characterization and interconversion of three forms of cholesterol oxidase extracted from Nocardia rhodochrous.

The physical properties and the methods used for interconversion of three forms of cholesterol oxidase extracted from Nocardia rhodochrous by treatment with Triton X-100, trypsin or buffer alone provide evidence that these forms differ chiefly in the possession or absence of a hydrophobic anchor region connected by a trypsin-sensitive region. The hydrophobic domain normally integrates the enzyme into the cell membrane and confers amphipathic properties on the solubilized enzyme, causing adsorption to hydrophobic resins, aggregation when detergent is removed and formation of mixed micelles with detergent and cholesterol resulting in surface-dilution kinetic behaviour and activation by relatively high concentrations of water-miscible solvents. By contrast, only the enzymic fragment is extracted with trypsin and it behaves as a conventional soluble enzyme and does not aggregate or interact with hydrophobic resins, detergents or water-miscible solvents. As no phospholipid could be detected in the enzyme extracts, the detergent appears to act as a substitute for the cell-membrane lipids that would normally interact with the hydrophobic region. This cholesterol oxidase is an example of a prokaryotic enzyme possessing two closely associated catalytic functions, dehydrogenase and isomerase activities, and an anchoring function.     

1H-NMR assignments and the dynamics of interconversion of the isomeric forms of cytochrome b5 in solution

Cytochrome b5 reconstituted with specifically deuterated hemins has led to the assignment of the resolved 6,7 beta-propionate protons and heme meso protons. Freshly reconstituted cytochrome b5 contains a mixture of two isomers in an approx. 1:1 ratio. As time proceeds the minor isomer decreases in intensity until the equilibrium ratio, approx. 8:1, of the two isomers is reached. The rate of the heme disorder kinetics was investigated for cytochrome b5 as a function of pH, oxidation state and 2,4 heme substitutents. Comparison of the kinetic data for cytochrome b5 with that obtained for other b-type heme proteins supports the proposal that the heme disorder arises from a 180 degree rotation of the heme about the alpha, gamma-meso axis. Computer-difference methods allow the spectra of the two individual isomers to be generated. Comparison of the NMR spectral parameters for the two individual isomers indicates small structural differences for amino acid side-chain orientations.     

Resonance Raman spectra of cytochrome c oxidase. Excitation in the 600-nm region.

The resonance Raman (RR) spectra of oxidized, reduced, and oxidized cyanide-bound cytochrome c oxidase with excitation at several wavelengths in the 600-nm region are presented. No evidence is found for laser-induced photoreduction of the oxidized protein with irradiation at lambda approximately 600 nm at 195 K, in contrast to the predominance of this process upon irradiation in the Soret region at this temperature. The Raman spectra of all three protein species are very similar, and there are no Raman bands which are readily assignable to either cytochrome a or cytochrome a3 exclusively. The Raman spectra of the three protein species do, however, exhibit a number of bands not observed in the RR spectra of other hemoproteins upon exicitation in their visible absorption bands. In particular, strong Raman bands are observed in the low-frequency region of the RR spectra (less than 500 cm-1). The frequencies of these bands are similar to those of the copper-ligand vibrations observed in the RR spectra of type 1 copper proteins upon excitation in the 600-nm absorption band characteristic of these proteins. In cytochrome c oxidase, these bands do not disappear upon reduction of the protein and, therefore, cannot be attributed to copper-ligand vibrations. Thus, all the observed RR bands are associated with the two heme A moieties in the enzyme.     

Electron transfer in monomeric forms of beef and shark heart cytochrome c oxidase

Beef heart cytochrome c oxidase is dimeric in reconstituted membranes and in nonionic detergents at physiological pH [Henderson, R., Capaldi, R. A., & Leigh, J. (1977) J. Mol. Biol. 112, 631; Robinson, N.C., & Capaldi, R. A. (1977) Biochemistry 16, 375], raising the possibility that this aggregation state is a prerequisite for enzymatic activity. A procedure for dissociating the enzyme into monomers is presented. This involves treating the protein with high concentrations of Triton X-100 at pH 8.5. The electron transfer activity of the monomer is comparable to that of the dimer under identical assay conditions. The beef heart cytochrome c oxidase monomer was found to be heterogeneous in hydrodynamic studies, probably due to dissociation of associated polypeptides, including subunit III. Monomer molecular weights in the range 129 000-160 000 were obtained. Previous studies have indicated that shark heart cytochrome c oxidase is monomeric under physiological conditions. Sedimentation equilibrium studies reported here confirm this. The elasmobranch enzyme, with a similar polypeptide composition to that of beef enzyme, was determined to have a molecular weight of 158 000.     

A comparison of the resonance Raman properties of the fast and slow forms of cytochrome oxidase

Resonance Raman (RR) spectra of the "rapid" and "slow" forms (Baker et al., 1987) of resting cytochrome oxidase obtained with Soret excitation at 413.1 nm are reported. There are a number of conspicuous differences between the two forms in the high-frequency region of the RR spectrum which involve changes in Raman intensity arising from a blue shift in the Soret maximum of cytochrome a3 upon conversion to the slow form. In the low-frequency region a peak present at 223 cm-1 in the rapid form shifts to 220 cm-1 in the slow form; this peak is assigned as the cytochrome a3 Fe(III)-N(His-Im) stretch. The slow form of the enzyme possesses greater intensity in RR peaks near 1620 cm-1 which have been previously attributed by others to partial photoreduction of the enzyme. We have quantitated the amount of laser-induced photoreduction in these RR spectra by comparison with the spectra of mixed-valence derivatives of the enzyme and find that these 1620-cm-1 features are unreliable indicators of photoreduction. The spectra of the fast- and slow-reacting species in H2O and D2O have been compared. The fast-reacting form exhibits a 4-cm-1 shift, from 223 to 219 cm-1, upon transferring to D2O in a peak which we assign as the cytochrome a3 Fe(III)-N(His-Im) stretch. There is a parallel shift in the feature at 1651 cm-1 due to the C = O stretch of the formyl group of cytochrome a. These deuterium shifts are not observed in the slow form.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple frequency EPR studies on three forms of oxidized cytochrome c oxidase

Bovine heart mitochondrial cytochrome c oxidase (cytochrome aa3) (EC 1.9.3.1) has been demonstrated to occur in several forms when the redox centers in the protein are thought to be fully oxidized. We report here the results of extensive EPR studies at 3, 8.9, 9.2, 9.4, 15 and 34 GHz on the resting state, the alternative resting state (with g = 12 at 9 GHz) and pulsed state (with g = 5 signal at 9 GHz). Theoretical consideration is given to all binary spin-coupling possibilities under the constraint that the iron atoms are either ferric or ferrous and the copper atoms are either cupric or cuprous. We conclude that the g = 12 signal can arise from any spin system with S greater than 1 and D = 0.15 cm-1. The g = 5 signals originate from an excited, integer-spin system with D = 0.035 cm-1, which is approximately 7 cm-1 above the ground state (not observed in EPR). It is pointed out that in interpretations of data and elaboration of suitable models in this field, the implications of spin-coupling should be considered in a comprehensive and not in a selective way. At 3 GHz, EPR spectra of CuA in the resting, pulsed and anaerobically oxidized states show that this center is identical in its EPR for all three states.     

Cytochrome c oxidase: Intermediates of the catalytic cycle and their energy-coupled interconversion

Several issues relevant to the current studies of cytochrome c oxidase catalytic mechanism are discussed. The following points are raised. (1) The terminology currently used to describe the catalytic cycle of cytochrome oxidase is outdated and rather confusing. Presumably, it would be revised so as to share nomenclature of the intermediates with other oxygen-reactive heme enzymes like P450 or peroxidases. (2) A "catalytic cycle" of cytochrome oxidase involving complete reduction of the enzyme by 4 electrons followed by oxidation by O(2) is a chimera composed artificially from two partial reactions, reductive and oxidative phases, that never operate together as a true multi-turnover catalytic cycle. The 4e(-) reduction-oxidation cycle would not serve a paradigm for oxygen reduction mechanism and protonmotive function of cytochrome oxidase. (3) The foremost role of the K-proton channel in the catalytic cycle may consist in securing faultless delivery of protons for heterolytic O-O bond cleavage in the oxygen-reducing site, minimizing the danger of homolytic scission reaction route. (4) Protonmotive mechanism of cytochrome oxidase may vary notably for the different single-electron steps in the catalytic cycle.     

Studies on cytochrome oxidase. Interactions of the cytochrome oxidase protein with phospholipids and cytochrome c.

1. By the application of the principle of the sequential fragmentation of the respiratory chain, a simple-method has been developed for the isolation of phospholipid-depleted and phospholipid-rich cytochrome oxidase preparations. 2. The phospholip-rich oxidase contains about 20% lipid, including mainly phosphatidylethanolamine, phosphatidylcholine, and cardiolipin. Its enzymic activity is not stimulated by an external lipid such as asolectin. 3. The phospholipid-depleted oxidase contains less than 0.1% lipid. It is enzymically inactive in catalyzing the oxidation of reduced cytochrome c by molecular oxygen. This activity can be fully restored by asolectin; and partially restored (approximately 75%) by purified phospholipids individually or in combination. The activity can be partially restored also by phospholipid mixtures isolated from mitochondria, from the oxidase itself, and from related preparations. Among the detergents tested only Emasol-1130 and Tween 80 show some stimulatory activity. 4. The phospholipid-depleted oxidase binds with cytochrome c evidently by "protein-protein" interactions as does the phospholipid-rich or the phospholipid-replenished oxidase to form a complex with the ratio of cytochrome c to heme a of unity. The complex prepared from phospholipid-depleted cytochrome oxidase exhibits a characteristic Soret absorption maximum at 415 nm in the difference spectrum of the carbon monoxide-reacted reduced form minus the reduced form. This 415-nm maximum is abolished by the replenishment of the complex with a phospholipid or by the dissociation of the complex in cholate or in a medium of high ionic strength. When ascorbate is used as an electron donor, the complex prepared from phospholipid-depleted cytochrome oxidase does not cause the reduction of cytochrome a3 which is in dramatic contrast to the complex from the phospholipid-rich or the phospholipid-replenished oxidase. However, dithionite reduces cytochrome a3 in all of the preparations of the cytochrome c-cytochrome oxidase complex. These facts suggest that the action of phospholipid on the electron transfer in cytochrome oxidase may be at the step between cytochromes a and a3. This conclusion is substantiated by preliminary kinetic results that the electron transfer from cytochrome a to a3 is much slower in the phospholipid-depleted than in phospholipid-rich or phospholipid-replenished oxidase. On the basis of the cytochrome c content, the enzymic activity has been found to be about 10 times higher in the system with the complex (in the presence of the replenishedhe external medium unless energy is provided, and that     

Low-spin ferric forms of cytochrome a3 in mixed-ligand and partially reduced cyanide-bound derivatives of cytochrome c oxidase.

Optical-absorption-, e.p.r.- and m.c.d. (magnetic-circular-dichroism)-spectroscopic measurements were made on liganded derivatives of oxidized and partially reduced cytochrome c oxidase. When NO was added to oxidized cyanide-bound cytochrome c oxidase, no changes occurred in the optical-absorption difference spectrum. In contrast, NO induced reduction of cytochrome a3 and formation of the nitrosylferrohaem species when the oxidized resting enzyme was the starting material. E.p.r. spectroscopy of the NO-treated oxidized cyanide-bound enzyme revealed the presence of a low-spin haem signal at g = 3.40, whereas the g = 3.02 and g = 2.0 signals of the oxidized enzyme remained unchanged. Both haem groups in this species are e.p.r.-detectable simultaneously. Examination of an identical sample by m.c.d. spectroscopy in the near-i.r. region identified two distinct low-spin species at 1565 and 1785 nm. Irradiation with white light of the NO-treated cyanide-bound sample at 10K resulted in the disappearance of the g = 3.40 e.p.r. signal and the m.c.d. signal at 1785 nm, whereas a band at 1950nm increased in intensity. When the photolysed sample was warmed to 50K and held in the dark for 15 min, the original spectrum returned. Magnetization studies of the 1785nm m.c.d. band support the assignment of this signal to the same metal centre that gives rise to the g = 3.40 e.p.r. signal. The effect of NO on the oxidized cyanide-bound enzyme was compared with that obtained when the oxidized cyanide-bound species was taken to the partially reduced state. Cytochrome a3 is e.p.r.-detectable with a g-value of 3.58 [Johnson, Eglinton, Gooding, Greenwood & Thomson (1981) Biochem. J. 193, 699-708]. Its near-i.r. m.c.d. spectrum shifts from 1950nm in the oxidized cyanide-bound enzyme to 1545nm on addition of reductant. A scheme is advanced for the structure of the cytochrome a3-CuB site that allows for cyanide binding to Fea3 and NO binding to CuB. Cyanide is the bridging ligand in the ferromagnetically coupled cytochrome a3-CuB pair of oxidized cyanide-bound cytochrome c oxidase. The bridged structure and the magnetic interaction are broken when the enzyme is partially reduced. However, when NO binds to CuB the cyanide bridge remains intact, but now the odd spins of NO and CuB are magnetically coupled.     

Coulometric and potentiometric evaluation of the redox components of cytochrome c oxidase in situ

A new coulometric-potentiometric titration cuvette is described which permits accurate measurements of oxidation-reduction components in membranous systems. This cuvette has been utilized to measure the properties of cytochrome c oxidase in intact membranes of pigeon breast muscle mitochondria. The reducing equivalents accepted and donated by the portion of the respiratory chain with half-reduction potentials greater than 200 mV are equal to those required for the known components (cytochrome a3 and the high-potential copper plus cytochrome a, 'visible copper', cytochrome c1, cytochrome c, and the Rieske iron-sulfur protein). Titrations in the presence of CO show that formation of the reduced cytochrome a3-CO complex requires two reducing equivalents per cytochrome a3 (coulometric titration). Potentiometric titrations indicate (Lindsay, J.G., Owen, C.S. and Wilson, D.F. (1975) Arch. Biochem. Biophys. 169, 492--505) that both cytochromes a3 and the high-potential copper must be reduced in order to form the CO complex (n = 2.0 with a CO concentration-dependent half-reduction potential, Em). By contrast, titrations in the presence of azide show that the Em value of the high-potential copper is unchanged by the presence of azide and thus azide binds with nearly equal affinity whether the copper is reduced or oxidized.     

Intermediate forms of cytochrome oxidase observed in transient kinetic experiments and those visited in the catalytic cycle

The cytochrome oxidase family of heme-copper oxidases has been the subject of intense kinetic and mechanistic enquiry. Much of this work has focussed on transient kinetic studies of the partial reactions of the enzyme with the goal being to build a kinetic model describing the catalytic cycle that the enzyme undergoes to direct the oxidation of substrate, reduction of oxygen and vectorial proton transfer. A key aspect of such a model is to define the structures of each of the intermediate forms the enzyme takes up as it traverses the catalytic cycle. One complication that has been prevalent with mitochondrial cytochrome c oxidase is the existence of structural variants of the enzyme, as isolated, that may not be participants in catalysis. Studies of structurally simpler procaryotic members of the family may offer new insight on the intermediates of catalysis. In this paper transient-state and steady-state kinetic studies of cytochrome aa(3)-600 from Bacillus subtilis are integrated into a model of the catalytic cycle. This model specifies that the P intermediate accumulates in the steady-state and it is proposed that the step following its formation is limited by proton uptake.     

Construction of histidine-tagged yeast mitochondrial cytochrome c oxidase for facile purification of mutant forms

Yeast CcO (cytochrome c oxidase) has been developed as a facile system for the production and analysis of mutants of a mitochondrial form of CcO for mechanistic studies. First, a 6H tag (His6 tag) was fused to the C-terminus of a nuclear-encoded subunit of CcO from yeast Saccharomyces cerevisiae. This allowed efficient purification of a WT (wild-type) mitochondrial CcO, 6H-WT (yeast CcO with a 6H tag on the nuclear-encoded Cox13 subunit), with a recovery yield of 45%. Its catalytic-centre activity [≈180 e·s(-1) (electrons per s)], UV-visible signatures of oxidized and reduced states and ability to form the P(M) ['peroxy' (but actually a ferryl/radical state)] and F (ferryl) intermediates confirm normal functioning of the histidine-tagged protein. Point mutations were introduced into subunit I of the 6H-WT strain. All mutants were screened for their ability to assemble CcO and grow on respiratory substrate. One such mutant [6H-E243DI (the 6H-WT strain with an additional mutation of E243D in mitochondrial DNA-encoded subunit I)] was purified and showed ~50% of the 6H-WT catalytic-centre activity, consistent with the effects of the equivalent mutation in bacterial oxidases. Mutations in both the D and the H channels affect respiratory growth and these effects are discussed in terms of their putative roles in CcO mechanism.     

The interactions between cytochrome c and cytochrome oxidase that determine the conformation of the oxidized oxidase.

1. Cytochrome c2+ increases the rate at which cytochrome oxidase (EC 1.9.3.1) gamma max428nm) converts to its conformational isomer (gamma max 418-423 nm) but cytochrome c3+ has little effect on the conversion rate. 2. Interactions between reduced cytochrome oxidase and cytochrome c were studied in the absence of electron flow using anaerobic Sephadex columns. 3. Oxidase that is reduced by cytochrome c2+ or other reductant forms the 418-to 423-nm isomer if its last contact, before oxidation, is with cytochrome c3+. If the reduced oxidase contacts cytochrome c2+, before oxidation, the 428-nm oxidase forms.