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.     

Current issues in the chemistry of cytochromec oxidase

Some contemporary issues relevant to the chemistry of mammalian cytochromec oxidase are discussed. These include the optical properties of heme A and the spectroscopic consequences of the differences in side-chain substitution compared to heme B; a common fallacy concerning the electrostatic exchange interaction between cytochromea ₃ and Cu_B; the question of the number and location of the copper components of the enzyme; and the mode of binding of ligands such as cyanide and azide.     

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.     

Succinylated bovine heart mitochondrial cytochromec oxidase

Cytochromec oxidase was prepared by sequential extraction of bovine heart muscle submitochondrial particles with sodium deoxycholate, followed by fractional precipitation with ammonium sulfate and chromatography on Sephadex G-75. The resulting preparation had typical absorption spectra, an activity of 1.28 sec^–1 (mg protein)^–1 (3 ml)^–1 in deoxycholate or 4.13 sec^–1 (mg protein)^–1 (3 ml)^–1 in 0.5% Tween 80, and a minimum molecular weight of 120,000 daltons as calculated from the heme content and the total protein. Amino acid analyses of nine preparations yielded a molecular weight per heme of 86,500 daltons. The net charge was calculated to be +8.7 at pH 7.0. Succinylation of cytochromec oxidase in the presence of 500 molar excess of succinic anhydride produced a soluble preparation having a negative charge at neutral pH. The modified enzyme was highly autoxidizable and had little or no activity toward ferrocytochromec as a substrate. Its averageS _20,w was 5.8 and its apparentD was 4.0 × 10^–7 cm² sec^–1, from which a molecular weight of 126,000 daltons was calculated. This size of enzyme is considered to be that of the monomer, because the value is practically the same as the minimum molecular weight reported herein, and since it is approximately onehalf the value obtained in our laboratory (and in others) for the unmodified enzyme.     

The kinetics of electron entry in cytochromec oxidase

Summary The kinetics of electron entry in beef heart cytochromec oxidase have been studied by stopped-flow spectroscopy following chemical modification of the Cu_A site with mercurials. In this derivative Cu_A is no longer reducible by cytochrome c while cytochromea may accept electrons from the latter with rates comparable to the native enzyme. The results indicate that Cu_A is not the exclusive electron entry site in cytochromec oxidase.     

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.     

Genes coding for cytochromec oxidase inParacoccus denitrificans

Several loci on theParacoccus denitrificans chromosome are involved in the synthesis of cytochromec oxidase. So far three genetic loci have been isolated. One of them contains the structural genes of subunits II and III, as well as two regulatory genes which probably code for oxidase-specific assembly factors. In addition, two distinct genes for subunit I have been cloned, one of which is located adjacent to the cytochromec ₅₅₀ gene. An alignment of six promoter regions reveals only short common sequences.     

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.     

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.     

Immunohistochemical localization of xanthine oxidase in human cardiac and skeletal muscle

The generation of a monoclonal antibody specific to xanthine oxidase and its use in the distribution of the enzyme in human tissue is described. Xanthine oxidase was purified from human and bovine milk by a rapid method, allowing for minimal proteolytic degradation, and the purified enzyme preparations were used for the immunization of BALB/c mice as well as for the subsequent selection of hybridomas. The hybridoma clone X1–7, IgG (2a, -light chain) was selected for further analysis and demonstrated to precipitate xanthine oxidase from human liver and skeletal muscle extracts. As determined by SDS-polyacrylamide gel electrophoresis of eluates from affinity chromatography, the X1–7 antibody bound to a main protein of 155 kDa, from human milk and skeletal muscle, and to proteins of 155, 143 and 95 kDa from human liver. Immunohistochemical studies, using two of the monoclonal antibodies with differing epitope specificity, revealed xanthine oxidase to be localized mainly in the vascular smooth muscle cells but also in a proportion of endothelial cells of capillaries and smaller vessels in both human cardiac and skeletal muscle. Immunoreactivity was additionally observed in human macrophages and mast cells. The results of the present study confirm previous reports of the presence of xanthine oxidase in capillary endothelial cells, but also demonstrates additional localization of the enzyme in vascular smooth muscle cells, macrophages and mast cells. The current findings verify that the distribution of xanthine oxidase in human tissue includes cardiac and skeletal muscle.     

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     

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.     

Kinetic studies of the lipid requirement of mitochondrial cytochromec oxidase

The lipid requirement of cytochromec oxidase was reinvestigated using both acetone and phospholipase A to deplete mitochondria of lipid. Removal of lipid resulted in a decrease in both the apparentK _m for cytochromec and apparentV _max when compared to control mitochondria. Addition of phospholipid to the assay mixture reactivated the enzyme. For both treatments theK _m returned to the control value. With phospholipase A treated mitochondria theV _max increased to near the control value, while acetone extracted mitochondria could be restored to aV _max of 1/2 that of the control. Detergent does not substitute for phospholipid and inhibits the reactivation with phospholipid.This research was supported in part by United States Public Health Service Research Grant AM-14632 and a Grant-in-Aid of the American Heart Association.     

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.     

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.     

The sequence of electron carriers in the reaction of cytochromec oxidase with oxygen

Kinetic studies of the electron transfer processes performed by cytochrome oxidase have assigned rates of electron transfer between the metal centers involved in the oxidation of ferrocytochromec by molecular oxygen. Transient-state studies of the reaction with oxygen have led to the proposal of a sequence of carriers from cytochromec, to Cu_A, to cytochromea, and then to the binuclear (i.e., cytochromea ₃-Cu_B) center. Electron exchange rates between these centers agree with relative center-to-center distances as follows; cytochromec to Cu_A 5–7 Å, cytochromec to cytochromea 20–25 Å, Cu_A to cytochromea 14–16 Å and cytochromea to cytochrome a₃-Cu_B 8–10 Å. It is proposed that the step from cytochromea to the binuclear center is the key control point in the reaction and that this step is one of the major points of energy transduction in the reaction cycle.     

Immunohistochemical demonstration of fibre type-specific isozymes of cytochrome c oxidase in human skeletal muscle

Summary The immunohistochemical reaction of monoclonal as well as polyclonal antibodies against cytochrome c oxidase (COX) subunits with serial sections of normal human skeletal muscle was investigated. The stronger reactivity of polyclonal antibodies to COX subunits II–III and VIIbc with type I as compared to type II fibres, correlated well with the higher histochemical reactivity of NADH dehydrogenase, succinate dehydrogenase and cytochrome c oxidase in type I fibres. In contrast an almost exclusive reaction of a monoclonal antibody against subunit IV with type I fibre and a preponderan reaction of a polyclonal antibody against subunits Vab with type II fibres was obtained. Antibodies against subuntis I, Vb and VIc did not reveal a fibre-type-specific reactivity. The data indicate in human muscle the occurrence of fibre type-specific isozymes of cytochrome c oxidase differing in subunits IV and Va or Vb.     

Immunohistochemical demonstration of fibre type-specific isozymes of cytochrome c oxidase in human skeletal muscle

The immunohistochemical reaction of monoclonal as well as polyclonal antibodies against cytochrome c oxidase (COX) subunits with serial sections of normal human skeletal muscle was investigated. The stronger reactivity of polyclonal antibodies to COX subunits II-III and VIIbc with type I as compared to type II fibres, correlated well with the higher histochemical reactivity of NADH dehydrogenase, succinate dehydrogenase and cytochrome c oxidase in type I fibres. In contrast an almost exclusive reaction of a monoclonal antibody against subunit IV with type I fibre and a preponderant reaction of a polyclonal antibody against subunits Vab with type II fibres was obtained. Antibodies against subunits I, Vb and VIc did not reveal a fibre-type-specific reactivity. The data indicate in human muscle the occurrence of fibre type-specific isozymes of cytochrome c oxidase differing in subunits IV and Va or Vb.     

Studies on the molecular basis of H+ translocation by cytochromec oxidase

We report here studies which characterize further the interaction ofN,N-dicyclohexylcarbodiimide with cytochromec oxidase leading to inhibition of H⁺ translocation by the enzyme. Further evidence is presented to show that the inhibition results from a real interaction of DCCD with the enzyme and cannot be accounted for by uncoupling and, contrary to recent criticisms, this interaction occurs specifically with subunit III of the enzyme even at relatively high inhibitor-to-enzyme stoichiometries. Use of a spin-label analogue of DCCD has enabled us to demonstrate that the carbodiimide-binding site is highly apolar and may not lie on the pathway of electron transfer.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - NCCD N-(2, 2, 6, 6-tetramethylpiperidyl-1-oxyl)-N-(cyclohexyl)carbodiimide - Hepes 2-(N-2-hydroxyethylpiperazin-N-yl) ethane sulfonate - TMPD N,N,N,N-tetramethylphenylenediamine