Preparation Of Cytochrome Oxidase Research Articles

The Kinetics of Enzyme Mixtures.

Even purified enzyme preparations are often heterogeneous. For example, preparations of aspartate aminotransferase or cytochrome oxidase can consist of several different forms of the enzyme. For this reason we consider how different the kinetics of the reactions catalysed by a mixture of forms of an enzyme must be to provide some indication of the characteristics of the species present. Based on the standard Michaelis-Menten model, we show that if the Michaelis constants (Km) of two isoforms differ by a factor of at least 20 the steady-state kinetics can be used to characterise the mixture. However, even if heterogeneity is reflected in the kinetic data, the proportions of the different forms of the enzyme cannot be estimated from the kinetic data alone. Consequently, the heterogeneity of enzyme preparations is rarely reflected in measurements of their steady-state kinetics unless the species present have significantly different kinetic properties. This has two implications: (1) it is difficult, but not impossible, to detect molecular heterogeneity using kinetic data and (2) even when it is possible, a considerable quantity of high quality data is required.

Projection structure of the cytochrome bo ubiquinol oxidase from Escherichia coli at 6 A resolution.

The haem-copper cytochrome oxidases are terminal catalysts of the respiratory chains in aerobic organisms. These integral membrane protein complexes catalyse the reduction of molecular oxygen to water and utilize the free energy of this reaction to generate a transmembrane proton gradient. Quinol oxidase complexes such as the Escherichia coli cytochrome bo belong to this superfamily. To elucidate the similarities as well as differences between ubiquinol and cytochrome c oxidases, we have analysed two-dimensional crystals of cytochrome bo by cryo-electron microscopy. The crystals diffract beyond 5 A. A projection map was calculated to a resolution of 6 A. All four subunits can be identified and single alpha-helices are resolved within the density for the protein complex. The comparison with the three-dimensional structure of cytochrome c oxidase shows the clear structural similarity within the common functional core surrounding the metal-binding sites in subunit I. It also indicates subtle differences which are due to the distinct subunit composition. This study can be extended to a three-dimensional structure analysis of the quinol oxidase complex by electron image processing of tilted crystals.

The mole nose instructs the brain

Star-nosed moles normally have 11 mobile appendages, or rays, extending from each side of the nose. In cytochrome oxidase preparations, each ray is visible in primary somatosensory cortex as a dark band separated by light septa. When a single mole was found with 12 rays on each side of the nose, primary somatosensory cortex had 12 bands. We take this as further evidence that morphological features of somatosensory cortex are determined by the periphery.

Structure and thermal denaturation of crystalline and noncrystalline cytochrome oxidase as studied by infrared spectroscopy

Fourier-transform infrared spectroscopy has been applied to the study of lipid vesicle-supported two-dimensional crystals and noncrystalline preparations of beef heart cytochrome oxidase. At room temperature, no conformational differences are seen between the noncrystalline and crystalline proteins, whose conformation is shown to consist of ca. 40% alpha-helix, 20% extended structures (including beta-sheet), 17% beta-turns, and 22% open loops plus nonstructured conformations. A novel infrared approach that combines quantitative spectral band decomposition with the study of the thermal behavior of each component has been applied. The procedure allows the independent examination of temperature-induced changes in individual structural elements (alpha-helix, beta-sheet, beta-turns, and unordered). All these reflect, upon heating the protein from 20 to 80 degrees C, a major irreversible thermal event centred at 55-60 degrees C, leading to a molecular state devoid of enzyme activity but with a defined secondary structure; in addition, when the band position, percent area (integrated intensity), and bandwidth of the various amide I components are separately plotted versus temperature, each component is seen to behave in a characteristic way. Thermal denaturation in D2O buffer shows a decrease in nonstructured conformations and an increase in beta-turns without major changes in the proportion of alpha-helix. Temperature-induced changes are not the same in amorphous and crystalline structures, the latter being in general more stable toward the thermal challenge. The above data extend and confirm previous structural studies on cytochrome oxidase using cryo-electron microscopy.

Reaction of formate with the fast form of cytochrome oxidase: a model for the fast to slow conversion.

The ability to isolate preparations of cytochrome oxidase which are highly homogeneous has facilitated a study of the effects of various reagents on the purified enzyme. The addition of either sodium formate, formamide, formaldehyde, or sodium nitrite to enzyme which reacts in a single rapid kinetic phase with cyanide causes a blue-shift of 4-6 nm of the net (cytochrome a + cytochrome a3) Soret maximum. Only the derivative prepared by adding sodium formate demonstrates measurable intensity in the g' = 12 region of the low-temperature electron paramagnetic resonance (EPR) spectrum. This g' = 12 resonance is characteristic of cytochrome oxidase which has undergone a modification at the binuclear center and thereby reacts sluggishly with cyanide. As the site of cyanide binding in resting enzyme as been demonstrated to be CuB [Yoshikawa, S., & Caughey, W.S. (1990) J. Biol. Chem. 265, 7945-7958], it is proposed that formate can bind to CuB and the fast to slow transition is rationalized by using this proposal. The g' = 12 signal is also produced upon the addition of sodium formate to mitochondrial preparations, suggesting that the species responsible for this behavior may have possible physiological relevance. Physical properties of the formate derivative and data for other reagents reacted with the fast-reacting enzyme preparation are presented.

Analytical characterization of cytochrome oxidase preparations with regard to metal and phospholipid contents, peptide composition and catalytic activity

A number of preparations of cytochrome oxidase have been analyzed for metals by energy dispersive X-ray fluorescence spectrometry. The EPR characteristics, the peptide compositions, the protein and phospholipid contents as well as the catalytic constants of the samples have also been determined. It is confirmed that the enzyme functional unit contains three copper atoms and one zinc atom in addition to two iron atoms. On the basis of the parameters determined for the different samples it is suggested that a high catalytic activity of a preparation can be correlated to a number of other analytical characteristics.

Fluorescein mercuric acetate specifically displaces zinc from cytochrome oxidase

Treatment of beef heart cytochrome oxidase with fluorescein mercuric acetate (FMA) was found to specifically displace zinc from the enzyme and inhibit the steady-state activity in a parallel fashion. The native cytochrome oxidase preparation contained 2.3 Cu : 2.0 Fe : 1.1 Zn : 0.9 Mg. Addition of 2 equivalents of FMA inhibited the activity by 50% and displaced 60% of the zinc from the enzyme, but did not affect the copper, iron or magnesium content. The pre-steady-state reduction of cytochrome oxidase by ferrocytochrome c was not affected by the FMA treatment, in contrast to the inhibition of steady state activity. These results suggest a possible structural or functional role for zinc in cytochrome oxidase.

Proton translocation by cytochrome oxidase vesicles catalyzing the peroxidatic oxidation of ferrocytochrome c.

Coupled with the peroxidatic oxidation of ferrocytochrome c under anaerobic conditions, proteoliposomes reconstituted with a purified preparation of bovine heart cytochrome oxidase ejected protons into the external medium with an apparent H+/e- ratio of 0.9. At the same time, protons in the intravesicular space were consumed. Dicyclohexylcarbodiimide significantly inhibited the proton translocation. Cyanide (0.14 mM) completely inhibited both the peroxidase and proton translocating activities. On the contrary, in the presence of 1 mM CO the proton ejection was abolished almost completely, but 50% of the peroxidase activity persisted. This result suggests the operation of multiple mechanisms in the peroxidase reaction and that the CO-sensitive one is coupled to the proton translocation.

Association of spin-labelled cardiolipin with dimyristoylphosphatidylcholine-substituted bovine heart cytochrome c oxidase. A generalized specificity increase rather than highly specific binding sites

The endogeneous lipid of bovine heart cytochrome c oxidase has been replaced by dimyristoylphosphatidylcholine using cholate-mediated exchange. The lipid-substituted preparation contained less than 1 mole cardiolipin per mole enzyme and possessed full oxidative activity. The association of spin-labelled cardiolipin with such lipid-substituted cytochrome oxidase preparations has been assayed using ESR spectroscopy. An average relative association constant 5.4-times that for phosphatidylcholine is obtained for cardiolipin. Measurements on preparations with increasing contents of unlabelled cardiolipin, introduced during lipid exchange, reveal that this selectivity corresponds to a generalized increase in specificity for all lipid association sites on the protein.

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.

Properties and reconstitution of a cytochrome oxidase deficient in subunit III.

Three different preparations of beef heart cytochrome oxidase (EC 1.9.3.1) were reconstituted into the membranes of artificial liposomes, and the electrical charge/electron ratios were determined for charge translocation coupled to enzymic activity. Our previously characterised subunit-III-deficient preparation, which apparently lacks H+ translocation capacity [Saraste et al. (1981) Eur. J. Biochem. 115, 261-268] has a decreased charge/electron ratio (0.9-1.0) as determined from the uptake of potassium in the presence of valinomycin, in contrast to the intact reconstituted cytochrome oxidase (1.9-2.0). A third preparation that was depleted of three minor polypeptides by trypsin treatment (these polypeptides are also removed together with subunit III using the present method), but which retains subunit III, had a K+/e- ratio of 1.5 but also a relatively low respiratory control index. The pH-dependence of the Em of cytochrome a determined in the presence of cyanide is abolished in the subunit-III-deficient enzyme. Electron transfer activities are nearly identical for the original and subunit-III-depleted enzymes at an infinite concentration of cytochrome c in a polarographic assay with supplemented phospholipids. The optical spectral properties are very similar for both preparations, but with a small shift to the blue of the alpha-peak in the modified enzyme. These results support the hypothesis that the removal of subunit III abolishes the H+-translocating function of cytochrome oxidase. This occurs by an intrinsic decoupling of H+ transport from electron transfer, and yields a preparation with only half-maximal efficiency of energy conservation.

Studies on the ferricytochrome a-ferrocytochrome a3-carbon monoxide complex of mammalian cytochrome oxidase. Conditions for preparation and some properties.

The conditions for the preparation of the ferricytochrome a-ferrocytochrome a3-carbon monoxide complex (a3+, a3(2)+CO) of cytochrome oxidase [EC 1.9.3.1] by the ferricyanide-reoxidation method and some properties of the prepared complex were studied. The addition of a small volume of concentrated ferricyanide solution to the dithionite-reduced and carbon monoxide-treated cytochrome oxidase preparation was required to obtain the (a3+, a3(2)+CO) spectrum showing absorption maxima at 590, 545, and 429 nm. The addition of larger volumes of ferricyanide solution, thus introducing larger amounts of oxygen into the preparation, caused decomposition of the carbon monoxide complex. A part of the added ferricyanide was immediately reduced by dithionite whereas the remainder was gradually reduced by partial oxidation product(s) of dithionite. The (a3+, a3(2)+CO) complex was stable only when excess ferricyanide remained in the reaction mixture. The formation of the (a3+, a3(2)+CO) spectrum was observed when sodium citrate, phosphate or borate buffer containing either cholate or a non-ionic detergent was employed as the solvent buffer, but not with the buffers containing sodium dodecyl sulfate (SDS) or cetyltrimethyl-ammonium bromide (CETAB). The formation was considerably inhibited by trishydroxymethyl-aminomethane(Tris)-HCl buffer. The (a3+, a3(2)+CO) spectrum appeared with maximal intensity at around pH 7. The pH-dependency of the intensity of the spectrum was not in parallel with the pH-dependent change of the polymerization state of the cytochrome oxidase preparation. On freezing to liquid nitrogen temperature, the (a3+, a3(2)+CO) complex prepared in usual solvent buffers was mostly converted to the oxidized form of cytochrome oxidase (a3+, a3(3)+. However, when prepared in the phosphate buffer, pH 8.0, containing 1.2% (w/v) sodium cholate and with 20% saturation with ammonium sulfate, the complex mostly remained unchanged after the freezing. Based on the results obtained, the stability of the juxta-heme structure of cytochrome a3 was also discussed.

Amino acid sequence of bovine heart cytochrome oxidase subunit IV (Albany)

The preliminary data on the amino acid sequence of subunit IV from bovine heart cytochrome oxidase (Albany) is presented. The subunit consists of 97 amino acids linked together in a single polypeptide chain. The sequence was established by the isolation, purification and sequencing of some of the tryptic, chymotryptic and thermolytic and Staphylococcus aureus protease peptides. This subunit is present in all cytochrome oxidase preparations. It corresponds to polypeptide VIa in cytochrome oxidase (Aachen) and subunit a in cytochrome oxidase (Eugene).

Carbon monoxide-binding cytochromes in the respiratory chain of the thermophilic bacterium PS3 grown with sufficient or limited aeration.

The effects of aeration on the growth and cytochrome patterns of thermophilic bacterium PS3 were studied; bacteria grown with strong aeration synthesized cytochromes c, b, and aa3, while those grown with low aeration, showing non-exponential growth, synthesized higher amounts of cytochromes c and b including o, and a lower amount of cytochrome a (a3). The CO-difference spectra indicated that the terminal oxidase was cytochrome aa3 for high aeration conditions and the cytochrome o for low aeration conditions. Cytochrome o can be solubilized by Triton X-100 from the membrane fraction of bacteria grown under oxygen-limited conditions. The carbon monoxide complex of cytochrome o, obtained by exposing this extract to CO, was photolyzed and the subsequent rebinding of CO was analyzed; it followed first order kinetics with a rate constant of around 8 s-1 at 25 degrees C. At liquid nitrogen temperature, CO-rebinding did not occur. The CO-difference spectrum of purified cytochrome oxidase sample from the bacteria grown with strong aeration (Sone, N., et al. (1979) FEBS Lett. 106, 39-42) revealed the presence of a small amount of a cytochrome o-like pigment besides cytochrome aa3. Analysis of the CO complexes of these chromophores showed rate constants of 29-30 s-1 for cytochrome aa3 and 35-42 s-1 for the o-like pigment, indicating that the cytochrome o-like pigment contaminating the purified cytochrome oxidase preparation was not typical cytochrome o.

A redox equilibrator for the preparation of cytochrome oxidase of mixed valence states and intermediate compounds for X-ray synchrotron studies

A redox titrator for the preparation of small volumes of highly concentrated biological samples for X-ray synchrotron studies provides for the redox equilibration at temperatures of 40 to −30°C. Anaerobic transfer directly to the X-ray sample chamber and mixing of an additional reagent just prior to freeze-trapping of the redox-equilibrated sample are provided.

Lipid--protein multiple binding equilibria in membranes.

Phospholipids at the lipid--protein interface of membrane proteins are in dynamic equilibrium with fluid bilayer. In order to express the number of binding sites (N) and the relative binding constants (K) in terms of measurable quantities, the equilibrium is formulated as an exchange reaction between lipid molecules competing for hydrophobic sites on the protein surface. Experimental data are reported on two integral membrane proteins, cytochrome oxidase and (Na,-K)-ATPase, reconstituted into defined phospholipids. Electron spin resonance measurements on reconstituted preparations of beef heart cytochrome oxidase in 1,2-dioleoyl-sn-3-phosphatidylcholine containing small quantities of the spin-labeled phospholipid 1-palmitoyl-2-(14-proxylstearoyl)-sn-3-phosphatidylcholine (PC*) gave a linear plot of bilayer/bound PC* vs. the lipid/protein ratio as predicted by the theory, with K congruent to 1 and N = 40 (normalized to heme aa3). This demonstrates that the spin-label moiety attached to the hydrocarbon chain does not significantly perturb the binding equilibria. In the second experimental system, (Na,K)-ATPase purified from rectal glands of Squalus acanthias was reconstituted with defined phosphatidylcholines as the lipid solvent and spin-labeled phospholipids with choline or serine head groups (PC*, PS*) as the solute. The (Na,K)-ATPase has a larger number of lipid binding or contact sites (N = 60-65 per alpha 2 beta 2 dimer) and exhibits a detectably larger average binding constant for the negatively charged phosphatidylserine than for the corresponding phosphatidylcholine. These results show that a multiple equilibria, noninteracting site binding treatment can account for the behavior of lipids exchanging between the protein surface and the lipid bilayer. Selective sites among a background of nonselective sites are experimentally detectable as a change in the measured relative binding constant.

An imidoester spin probe of membrane protein interactions: Application to cytochrome c

The synthesis of an imidoester spin label, whose advantages relative to other spin labels include its water solubility, lysine specificity, and retention of positive charge at the reaction site is described. Cytochrome c is spin labeled and shown to exhibit spectral changes upon interacting with lipid vesicles and lipid-rich cytochrome oxidase preparations. Spin labeled cytochrome c in buffer or in the presence of mitochondria at high ionic strength had a correlation time of τ = 0.91 ± 10 −9 s; at low ionic strength the mitochondrial signal was more immobilized, τ = 2.27 ± 0.13 × 10 −9 s; and further immobilization was observed when cytochrome c was bound to the high-affinity site of purified oxidase containing 37% phospholipid (τ = 2.71 ± 0.22 × 10 −9). Cytochrome c-oxidase electron transfer rates were unaltered by spin labeling. The results suggest that this imidoester spin label will be useful for studies of protein-protein and protein-lipid interactions.

Protein rotational mobility and lipid fluidity of purified and reconstituted cytochrome c oxidase.

The rotational mobility of spin-labeled bovine heart mitochondrial cytochrome c oxidase in purified form, and incorporated into lipid vesicles was studied. A rigidly attached short chain maleimide spin label permitted mobility by saturation transfer electron paramagnetic resonance. A long chain maleimide spin label was used to detect the fluidity of the lipid hydrocarbon region adjacent to the protein by conventional EPR. One method of preparing the purified enzyme resulted in a high degree of protein rotational mobility at 4/sup 0/C both in the purified detergent-solubilized enzyme (effective rotational correlation time of 100 ns) and in reconstituted membranes (correlation time of 40 ..mu..s). By contrast, another purification procedure resulted in little or no submillisecond protein rotational mobility both in purified form and in reconstituted membranes, suggesting the presence of large protein aggregates. Thus, the state of aggregation of cytochrome oxidase in membranes appears to depend on the state of aggregation prior to reconstitution. The mobile and immobile enzymes had the same high activity. In both reconstituted preparations, the bulk of the lipid was quite fluid at 4/sup 0/C, as probed by a free fatty acid spin label. The lipid hydrocarbon region adjacent to the protein, as probed by the long chain more » maleimide spin label, was also quite fluid in the membranes containing mobile enzyme,but was strongly immobilized in the membranes containing immobile enzyme. Thus, the strong immobilization of lipid in our preparations of cytochrome oxidase is apparently caused by protein-protein interactions, not by rigidity at the protein-lipid boundary. « less

Effect of ifenprodil on mitochondrial respiration of guinea pig brain

The effects of ifenprodil on respiratory activity in both the presence and absence of cyanide were examined using guinea pig brain mitochondria and mouse brain cytochrome oxidase preparations. Ifenprodil stimulated respiratory activity, exhibiting maximal effect at a concentration of 0.8 m m in the presence of succinate and potassium phosphate ( P i) among the various substrates, anions, and phosphonate compounds tested. The stimulatory action of ifenprodil on respiration could not be observed with frozen and thawed brain mitochondria. The stimulatory action was not affected by oligomycin but was reduced markedly by mersalyl. Dinitrophenol and flufenamic acid did not require the presence of P i for their stimulatory action on respiration. Ifenprodil abolished completely the cyanide-induced inhibition of respiration with succinate as a substrate, but elicited no significant effects against the cyanide-induced inhibition of cytochrome oxidase activity in both in vivo and in vitro experiments. The results suggest that ifenprodil seems to act on the mitochondrial membrane by accelerating P i and succinic acid transport through the membrane as well as by stimulating succinate oxidation, and that these stimulatory actions of ifenprodil may be involved in its protective effects against cyanide toxicity.

Resolution of cytochrome oxidase into two component complexes.

Cytochrome c oxidase (ferrocytochrome c: oxygen oxidoreductase, EC 1.9.3.1) has been resolved into a pair of complexes of unequal molecular weight. The larger complex (electron transfer complex) contains exclusively the oxidation-reduction proteins characteristic of cytochrome oxidase; the smaller complex (ion transfer complex) shows exclusively the capability for cation-dependent induction of the fluorescence of 8-anilino-1-naphthalenesulfonic acid--a capability demonstrable in preparations of cytochrome oxidase. The duplex nature of cytochrome oxidase has important implications for the mechanism of energy coupling.