Evolutionary aspects of cytochromec oxidase

The presence of additional subunits in cytochrome oxidase distinguish the multicellular eukaryotic enzyme from that of a simple unicellular bacterial enzyme. The number of these additional subunits increases with increasing evolutionary stage of the organism. Subunits I–III of the eukaryotic enzyme are related to the three bacterial subunits, and they are encoded on mito-chondrial DNA. The additional subunits are nuclear encoded. Experimental evidences are presented here to indicate that the lower enzymatic activity of the mammalian enzyme is due to the presence of nuclear-coded subunits. Dissociation of some of the nuclear-coded subunits (e.g., VIa) by laurylmaltoside and anions increased the activity of the rat liver enzyme to a value similar to that of the bacterial enzyme. Further, it is shown that the intraliposomal nucleotides influence the kinetics of ferrocytochromec oxidation by the reconstituted enzyme from bovine heart but not fromP. denitrificans. The regulatory function attributed to the nuclear-coded subunits of mammalian cytochromec oxidase is also demonstrated by the tissue-specific response of the reconstituted enzyme from bovine heart but not from bovine liver to intraliposomal ADP. These enzymes from bovine heart and liver differ in the amino acid sequences of subunits VIa, VIIa, and VIII. The results presented here are taken to indicate a regulation of cytochromec oxidase activity by nuclear-coded subunits which act like receptors for allosteric effectors and influence the catalytic activity of the core enzyme via conformational changes.     

Effect of trypsin on the kinetic properties of reconstituted beef heart cytochromec oxidase

Isolated beef heart cytochromec oxidase was reconstituted in liposomes by the cholate dialysis method with 85% of the binding site for cytochromec oriented to the outside. Trypsin cleaved specifically subunit VIa and half of subunit IV from the reconstituted enzyme. The kinetic properties of the reconstituted enzyme were changed by trypsin treatment if measured by the spectrophotometric assay but not by the polarographic assay. It is concluded that subunit VIa and/or subunit IV participate in the electron transport activity of cytochromec oxidase.     

Inhibition of the phosphate-stimulated cytochromec oxidase activity by thiophosphate

Yeast and mammalian cytochromec oxidase activity is inhibited by thiophosphate. This inhibition was observed when using either whole mitochondria or the isolated or reconstituted enzyme. The kinetics of the reduction reaction enabled us to demonstrate that thiophosphate acted on th electrons transfer between hemesa anda ₃. With whole mitochondria, phosphate alone stimulated respiration. The inhibition induced by thiophosphate was suppressed by phosphate only in mitochondria, but not when the isolated enzyme was used. The possibility of a kinetic regulation is discussed.Abbreviations CCCP p-carbonylcyanidem-chlorophenylhydrazone - TMPD N,N,N,N-tetramethylp-phenylenediamine - SPi thiophosphate     

Morphology of proteoliposomes containing fluorescein-phosphatidylethanolamine reconstituted with native and subunit III-depleted cytochromec oxidase

Beef heart cytochromec oxidase was reconstituted in asolectin liposomes containing the pH indicator fluorescein-phosphatidylethanolamine (FPE) by the cholate-dialysis procedure. The influence of PFE on the asolectin liposome size and of the removal of subunit III from the complex on its incorporation into liposomes was analyzed by freeze-fracture electron microscopy. Samples were frozen without the addition of cryoprotectants. The vesicle size distribution of native enzyme reconstituted into asolectin liposomes was homogenous, 84% of the population having a diameter of 14–37 ± 7.5 mm. The preparation containing FPE had a similar vesicle size distribution, but with bigger diameter range (20–50 nm). In all three different types of proteoliposome preparations the majority of particles containing vesicles was found to have 1 particle (42–81%). The absence of subunit III did not influence the incorporation of the enzyme into the liposomes and was as good as the preparation with native enzyme (>99%). Therefore we conclude that the suppression of the proton pump activity was due to the intrinsic properties of subunit III and not to defective incorporation into artificial membrane systems.Dedicated to the memory of Dr. R. P. Casey.     

Ferricytochromec induces monophasic kinetics of ferrocytochromec oxidation in cytochromec oxidase

The kinetics of ferrocytochromec oxidation by reconstituted cytochromec oxidase (COX) from bovine heart was followed by a spectrophotometric method, using on-line data collection and subsequent calculation of reaction rates from a function fitted to the progress curve. When reaction rates were calculated at increasing reaction times, the multiphasic kinetics of ferrocytochromec oxidation gradually changed into monophasic Michaelis-Menten kinetics. The same phenomenon was observed when ferrocytochromec oxidation was followed in the presence of increasing amounts of ferricytochromec. From these results we conclude that ferricytochromec shifts the multiphasic kinetics of ferrocytochromec oxidation by COX into monophasic kinetics, comparable to high ionic strength conditions. Furthermore, we show that ferricytochromec inhibits the high affinity phase of ferrocytochromec oxidation in an apparently competitive way, while inhibition of the low affinity phase is noncompetitive. These findings are consistent with a regulatory site model where both the catalytic and the regulatory site bind ferro- as well as ferricytochromec.     

Isolation and characterization of human heart cytochromec oxidase

Cytochromec oxidase was isolated from human hearts and separated by SDS gel electrophoresis. The identity of polypeptide bands with known subunits was demonstrated by immunoblotting with monospecific antisera to rat liver cytochromec oxidase subunits. The polarographically determined kinetics of cytochromec oxidation were similar to those reported for the bovine heart enzyme.     

The cytochromec oxidase from the yeastCandida parapsilosis

In the yeastCandida parapsilosis, the proteins encoded by mitochondrial DNA are different in number and size from those ofSaccharomyces cerevisiae. Nevertheless, the purified cytochromec oxidase fromCandida parapsilosis shows kinetic properties similar to those ofSaccharomyces cerevisiae.     

The K+-ionophores nonactin and valinomycin interact differently with the protein of reconstituted cytochromec oxidase

The K⁺-ionophores valinomycin and nonactin induce a qualitatively identical change of the visible spectrum of isolated oxidized cytochromec oxidase (red shift), but the amplitude is half with nonactin. Valinomycin, in the presence or absence of a protonophore, stimulates the respiration of the reconstituted enzyme to a higher extent than nonactin and results in a higherK _m for cytochromec. In contrast, nonactin causes a fivefold rate of proton conductivity across a liposomal membrane, after induction of a K⁺-diffusion potential. The data indicate that respiratory control by these antibiotics is not only due to degradation of a membrane potential, but rather to specific interaction with and modification of cytochromec oxidase.     

Probing heart cytochromec oxidase structure and function by infrared spectroscopy

IR spectra directly probe specific vibrators in bovine heart cytochromec oxidase, yielding quantitative as well as qualitative information on structures and reactions at these vibrators. C-O IR spectra reveal that CO binds to as two conformers each in isolated immobile environments sensitive to Fe _a and/or Cu_A oxidation state but remarkably insensitive to pH, medium, anesthetics, and other factors that affect activity. C-N IR spectra reveal that the one CN^– that binds to fully and partially oxidized enzyme can be in three different structures. These structures vary in relative amounts with redox level, thereby reflecting dynamic electron exchange among Fe _a , Cu_A, and Cu_B with associated changes in protein conformation of likely significance in O₂ reduction and H⁺-pumping. Azide IR spectra also reflect redox-dependent long-range effects. The amide I IR bands, due to C-O vibrators of peptide linkages and composed of multiple bands derived from different secondary structures, reveal high levels of -helix (60%) and subtle changes with redox level and exposure to anesthetics. N₂O IR spectra reveal that these anesthetic molecules at clinically relevant levels occupy three sites of different polarity within the enzyme as the enzyme is reversibly, but only partially, inhibited.     

Functional binding of cardiolipin to cytochromec oxidase

Bovine cytochromec oxidase usually contains 3–4 mol of tightly bound cardiolipin per cytochromeaa ₃ complex. At least two of these cardiolipins are required for full electron transport activity. Without the tightly bound cardiolipin, cytochromec oxidase has only 40–50% of its original activity when assayed in detergents that support activity, e.g., dodecyl maltoside. By measuring the restoration of electron transport activity, functional binding constants for cardiolipin and a number of cardiolipin analogues have been evaluated (K _d,app=1 µM for cardiolipin). These binding constants agree reasonably well with direct measurement of the binding using [¹⁴C]-acetyl-cardiolipin (K _d <0.1 µM) when the enzyme is solubilized with Triton X-100. These data are discussed in relationship to the wealth of data that is known about the association of cardiolipin with cytochromec oxidase and the other mitochrondrial electron transport complexes and transporters.     

The structure of cytochromec and the rates of molecular evolution

Summary The x-ray structure analysis of ferricytochromec shows the reasons for the evolutionary conservatism of hydrophobic and aromatic side chains, lysines, and glycines, which had been observed from comparisons of amino acid sequences from over 30 species. It also shows that the negative character of one portion of the molecular surface is conserved, even though individual acidic side chains are not, and that positive charges are localized around two hydrophobic channels leading from the interior to the surface.The reason for the unusual evolutionary conservation of surface features in cytochromesc is probably the interaction of the molecule with two other large macromolecular complexes, its reductase and oxidase. This conservation of surface structure also explains the relatively slow rate of change of cytochromec sequences in comparison with the globins and enzymes of similar size.The rate of evolution of a protein is the rate of occurrence of mutations in the genome modified by the probability that a random change in amino acid sequence will be tolerable in a functioning protein. The observed rates of change in fibrinopeptides, the globins, cytochromec, and several enzymes are interpreted in terms of the proteins' biological roles.Contribution No. 4114 from the Norman W. Church Laboratory of Chemical Biology, California Institute of Technology, Pasadena, California 91109.     

Activation studies by phospholipids on the purified cytochromec 4:o oxidase ofAzotobacter vinelandii

A modified procedure is described that was used to solubilize and purify the TMPD-dependent cytochromec ₄:o oxidase fromAzotobacter vinelandii. Two functional components (Fractions I and V) were obtained after DEAE-cellulose chromatography. Fraction V contained both cytochromec ₄ (3.6 nmol/mg protein) and cytochromeo (1.6 nmol/mg protein). This cytochrome oxidase complex oxidized TMPD at moderate rates. Fraction I, a clear greenish-yellow fraction, contained primarily phosphatidylethanolamine with some phosphatidylglycerol. Fraction I itself could not oxidize TMPD, but when it was preincubated with Fraction V, a 2–4-fold stimulation in TMPD oxidase activity occurred. Other authentic micellar phospholipids also readily activited TMPD oxidase activity in Fraction V. Themaximum activation effect obtained with Fraction I was in essence duplicated with purified phosphatidylethanolamine.Dedicated to the memory of David E. Green, a fine gentleman, an excellent scientist, and a true scholar. He will be missed by many of his former colleagues.     

The cytochromec reductase/oxidase respiratory pathway ofParacoccus denitrificans: Genetic and functional studies

Data are presented on three components of the quinol oxidation branch of theParacoccus respiratory chain: cytochromec reductase, cytochromec ₅₅₂, and thea-type terminal oxidase. Deletion mutants in thebc ₁ and theaa ₃ complex give insight into electron pathways, assembly processes, and stability of both redox complexes, and, moreover, are an important prerequisite for future site-directed mutagenesis experiments. In addition, evidence for a role of cytochromec ₅₅₂ in electron transport between complex III and IV is presented.     

On the role of subunit III in proton translocation in cytochromec oxidase

Mammalian mitochondrial cytochromec oxidase catalyzes the transfer of electrons from ferrocytochromec to molecular oxygen in the respiratory chain, while conserving the energy released during its electron transfer reactions by the vectorial movement of protons across the inner membrane of the mitochondrion. The protein domain that translocates the protons across the membrane is currently unknown. Recent research efforts have investigated the role of one of the transmembrane subunits of the enzyme (III,M _r 29,884) in the vectorial proton translocation reaction. The data that favor subunit III as integral in vectorial proton translocation as well as the data that support a more peripheral role for subunit III in proton translocation are reviewed. Possible experimental approaches to clarify this issue are presented and a general model discussed.     

Phylogenetic analysis of the perissodactylan family Tapiridae using mitochondrial cytochromec oxidase (COII) sequences

The four extant members of the family Tapiridae have a disjunct, relictual distribution, with three species being Neotropical (Tapirus bairdii, T. terrestris, andT. pinchaque) and one found in Southeast Asia (T. indicus). Little recent work on tapir systematics have appeared, and no molecular studies of this group have been published. A phylogenetic analysis was undertaken using sequences of the mitochondrial cytochromec oxidase subunit II gene (COII) from representatives of the four species of tapirs, as well as a representative outgroup,Equus caballus. Analyses of the COII sequences indicate a close relationship between the two South American species of tapirs,T. terrestris andT. pinchaque, and estimates of divergence dates using rates of COII evolution are compatible with migration of a single tapir lineage into South America following the emergence of the isthmus of Panama, about 3 million years bp. Various methods of analysis, including maximum parsimony, maximum likelihood, and neighbor-joining, provided poorer resolution of other tapir relationship. The COII data suggest that three distinct tapir mitochondrial lineages, a South American (represented byT. terrestris andT. pinchaque), a Central American (represented byT. bairdii), and an Asian (represented byT. indicus) diverged relatively rapidly, 20–30 million years bp. Another goal of this study was to calibrate the rate of COII evolution in a eutherian mammal group which has a good fossil record, such as perissodactyls, to estimate accurately the rate of COII evolution in a nonprimate mammalian group. The rate of COII evolution in equids and tapirs has been relatively constant and, using corrected distances, calibrated to be approximately 0.22% lineage/million years. This rate is three-to fourfold lower than that of hominoid primates.     

Cytochromec oxidase fromParacoccus denitrificans in Triton X-100: Aggregation state and kinetics

Cytochromec oxidase fromParacoccus denitrificans was homogenously dispersed in Triton X-100. Using gel exclusion chromatography and sucrose gradient centrifugation analysis a molecular weight of the detergent-protein complex of 155,000 was determined. After subtraction of the bound detergent (111 mol/mol hemeaa ₃) a molecular weight of 85,000 resulted, which agreed well with the model of a monomer containing two subunits. This monomer showed high cytochromec oxidase activity when measured spectrophotometrically in the presence of Triton X-100 (V _max=85 s^–1). The molecular activity, plotted according to Eadie-Hofstee, was monophasic as a function of the cytochromec concentration. AK _m of 3.6×10^–6 M was evaluated, similar to theK _m observed in the presence of dodecyl maltoside [Naeczet al. (1985).Biochim. Biophys. Acta 808, 259–272].     

Biosynthesis of superoxide dismutase and catalase inSaccharomyces cerevisiae: effects of oxygen and cytochromec deficiency

Summary Two strains ofSaccharomyces cerevisiae were used to study the synthesis of superoxide dismutase. One strain (cytochromec-deficient) contained 5–10% of the normal amounts of total cytochromec, while the other strain was a wild type. The cytochromec-deficient mutant had lower specific growth rate, growth yield, and oxygen uptake than the wild type. The superoxide dismutase and catalase activities, in both strains, were significantly lower under anaerobic than under aerobic conditions. Furthermore, under aerobic conditions the mutant contained higher levels of superoxide dismutase than the wild type which may be attributed to the higher intracellular flux of superoxide radicals caused by the cytochromec deficiency. The mutant also showed a lower level of catalase which was due to glucose repression.Paper Number 10007 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695, U.S.A. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of the products named, nor criticism of similar ones not mentioned.     

Interaction of horse cytochromec with the photosynthetic reaction center ofRhodospirillum rubrum

Mitochondrial cytochromec (horse), which is a very efficient electron donor to bacterial photosynthetic reaction centersin vitro, binds to the reaction center ofRhodospirillum rubrum with an approximate dissociation constant of 0.3–0.5 µM at pH 8.2 and low ionic strength. The binding site for the reaction center is on the frontside of cytochromec which is the side with the exposed heme edge, as revealed by differential chemical acetylation of lysines of free and reaction-center-bound cytochromec. In contrast, bacterial cytochromec ₂ was found previously to bind to the detergent-solubilized reaction center through its backside, i.e., the side opposite to the heme cleft [Rieder, R., Wiemken, V., Bachofen, R., and Bosshard, H. R. (1985).Biochem. Biophys. Res. Commun. 128, 120–126]. Binding of mitochondrial cytochromec but not of mitochondrial cytochromec ₂ is strongly inhibited by low concentrations of poly-l-lysine. The results are difficult to reconcile with the existence of an electron transfer site on the backside of cytochromec ₂.     

Experimental and theoretical analysis of the interaction between cytochromec and cytochromeb 5

Experimental and theoretical investigation of the interaction of cytochromec and cytochromeb ₅ performed over nearly twenty years has produced considerable insight into the manner in which these proteins recognize and bind to each other. The results of these studies and the experimental and theoretical strategies that have been developed to achieve these results have significant implications for understanding the behavior of similar complexes formed by more complex and less-well characterized electron transfer proteins. The current review provides a comprehensive summary and critical evaluation of the literature on which the current status of our understanding of the interaction of cytochromec and cytochromeb ₅ is based. The general issues related to the study of electron transfer complexes of this type are discussed and some new directions for future investigation of such systems are considered.     

Determination of reduction potential of an engineered Cu<Subscript>A</Subscript> azurin by cyclic voltammetry and spectrochemical titrations

The reduction potentials of an engineered Cu_A azurin in its native and thermally denatured states have been determined using cyclic voltammetry and spectrochemical titrations. Using a 4,4-dipyridyl disulfide modified gold electrode, the reduction potentials of native and thermally denatured Cu_A azurin are the same within the experimental error (422±5 and 425±5 mV vs. NHE, respectively, in 50 mM ammonium acetate buffer, pH 5.1, 300 mM NaCl, 25 °C), indicating that the potential is that of a nonnative state. In contrast, using a didodecyldimethylammonium bromide (DDAB) film-pyrolytic graphite edge (PGE) electrode, the reduction potentials of native and thermally denatured Cu_A azurin have been determined to be 271±7 mV (50 mM ammonium acetate buffer, pH 5.1, 4 °C) and 420±1 mV (50 mM ammonium acetate buffer, pH 5.1, 25 °C), respectively. Spectroscopic redox titration using [Ru(NH₃)₅Py]²⁺ resulted in a reduction potential (254±4 mV) (50 mM ammonium acetate buffer, pH 5.1, 4 °C) similar to the value obtained using the DDAB film-PGE electrochemical method. Complete reoxidation of [Ru(NH₃)₅Py]²⁺-reduced Cu_A azurin is also consistent with the conclusion that this spectrochemical titration method using [Ru(NH₃)₅Py]²⁺ measures the reduction potential of native Cu_A azurin.Abbreviations CcO cytochrome c oxidase - N₂OR nitrous oxide reductase - ET electron transfer - CV cyclic voltammetry - NHE normal hydrogen electrode - DDAB didodecyldimethylammonium bromide - PGE pyrolytic graphite edge