Structure and evolution of cytochrome oxidase

The structural features of cytochrome oxidases are reviewed in light of their evolution. The substrate specificity (quinol vs. cytochromec) is reflected in the presence of a unique copper centre (Cu _A ) in cytochromec oxidases. In several lines of evolution, quinol oxidases have independently lost this copper. Also, the most primitive cytochromec oxidases do not contain this copper, and electron entry takes place viac-type haems. These enzymes, exemplified by the rhizobial FixN complex, probably remind the first oxidases. They are related to the denitrification enzyme nitric oxide reductase.     

Protons,pumps, and potentials: Control of cytochrome oxidase

Cytochromec oxidase oxidizes cytochromec and reduces molecular oxygen to water. When the enzyme is embedded across a membrane, this process generates electrical and pH gradients, and these gradients inhibit enzyme turnover. This respiratory control process is seen both in intact mitochondria and in reconstituted proteoliposomes. Generation of pH gradients and their role in respiratory control are described. Both electron and proton movement seem to be implicated. A topochemical arrangement of redox centers, like that in the photosynthetic reaction center and the cytochromebc ₁ complex, ensures charge separation as a result of electron movement. Proton translocation does not require such a topology, although it does require alternating access to the two sides of the membrane by proton-donating and accepting groups. The sites of respiratory control within the enzyme are discussed and a model presented for electron transfer and proton pumping by the oxidase in the light of current knowledge of the transmembranous location of the redox centers involved.     

A structural model for the adduct between cytochrome c and cytochrome c oxidase

An ensemble of structural models of the adduct between cytochrome c and cytochrome c oxidase from Paracoccus denitrificans has been calculated based on the experimental data from site-directed mutagenesis and NMR experiments that have accumulated over the last years of research on this system. The residues from each protein that are at the protein–protein interface have been identified by the above experimental work, and this information has been converted in a series of restraints explicitly used in calculations. It is found that a single static structural model cannot satisfy all experimental data simultaneously. Therefore, it is proposed that the adduct exists as a dynamic ensemble of different orientations in equilibrium, and may be represented by a combination or average of the various limiting conformations calculated here. The equilibrium involves both conformations that are competent for electron transfer and conformations that are not. Long-range recognition of the partners is driven by non-specific electrostatic interactions, while at shorter distances hydrophobic contacts tune the reciprocal orientation. Electron transfer from cytochrome bc ₁ to cytochrome c oxidase is mediated through cytochrome c experiencing multiple encounters with both of its partners, only part of which are productive. The number of encounters, and thus the electron transfer rate, may be increased by the formation of a cytochrome bc ₁–cytochrome c oxidase supercomplex and/or (in human) by increasing the concentration of the two enzymes in the membrane space. Protein Data Bank Accession numbers The coordinates of the five best structural models for each of the four clusters have been deposited in the Protein Data Bank (PDB ID 1ZYY).     

Photoacoustic characterization of protein dynamics following CO photodetachment from fully reduced bovine cytochrome c oxidase

We report a protein conformational change following carbon monoxide photodetachment from fully reduced bovine cytochrome c oxidase that is hypothesized to be associated with changes in ligand mobility through a dioxygen access channel in the protein. Although not resolved by earlier photoacoustic or optical studies on this adduct, utilization of slightly lower temperatures revealed a process with a kinetic lifetime of about 70 ns at 10 degrees C. We measure an enthalpy change of about 8 kcal/mol in 0.050 M HEPES buffer that becomes less endothermic (DeltaH approximately 2 kcal/mol) at higher ionic strength. The volume contraction of about -0.7 mL/mol associated with the process almost doubles in higher ionic strength buffer systems. Measurements of samples in phosphate buffer systems are similar and appear to display the same subtle ionic strength dependence. Both the isolation of this photoacoustic signal component and the possible dependence on ionic strength of the thermodynamic parameters derived from its analysis appear analogous to and consistent with prior photoacoustic results monitoring CO photodetachment from the camphor complex of cytochrome P-450. Accordingly, we consider a similar model in which a conformational change results in movement of an exposed charged group or groups towards the interior of the protein, out of contact with solvent, as in the closing of a salt bridge.     

Evolution of the primate cytochrome c oxidase subunit II gene

We examined the nucleotide and amino acid sequence variation of the cytochrome c oxidase subunit II (COII) gene from 25 primates (4 hominoids, 8 Old World monkeys, 2 New World monkeys, 2 tarsiers, 7 lemuriforms, 2 lorisiforms). Marginal support was found for three phylogenetic conclusions: (1) sister-group relationship between tarsiers and a monkey/ape clade, (2) placement of the aye-aye (Daubentonia) sister to all other strepsirhine primates, and (3) rejection of a sister-group relationship of dwarf lemurs (i.e., Cheirogaleus) with lorisiform primates. Stronger support was found for a sister-group relationship between the ring-tail lemur (Lemur catta) and the gentle lemurs (Hapalemur). In congruence with previous studies on COII, we found that the monkeys and apes have undergone a nearly two-fold increase in the rate of amino acid replacement relative to other primates. Although functionally important amino acids are generally conserved among all primates, the acceleration in amino acid replacements in higher primates is associated with increased variation in the amino terminal end of the protein. Additionally, the replacement of two carboxyl-bearing residues (glutamate and aspartate) at positions 114 and 115 may provide a partial explanation for the poor enzyme kinetics in cross-reactions between the cytochromes c and cytochrome c oxidases of higher primates and other mammals. Correspondence to: R.L. Honeycutt     

Mammalian mitochondrial DNA evolution: A comparison of the cytochrome b and cytochrome c oxidase II genes

The evolution of two mitochondrial genes, cytochrome b and cytochrome c oxidase subunit II, was examined in several eutherian mammal orders, with special emphasis on the orders Artiodactyla and Rodentia. When analyzed using both maximum parsimony, with either equal or unequal character weighting, and neighbor joining, neither gene performed with a high degree of consistency in terms of the phylogenetic hypotheses supported. The phylogenetic inconsistencies observed for both these genes may be the result of several factors including differences in the rate of nucleotide substitution among particular lineages (especially between orders), base composition bias, transition/transversion bias, differences in codon usage, and different constraints and levels of homoplasy associated with first, second, and third codon positions. We discuss the implications of these findings for the molecular systematics of mammals, especially as they relate to recent hypotheses concerning the polyphyly of the order Rodentia, relationships among the Artiodactyla, and various interordinal relationships.Correspondence to: R.L. Honeycutt     

The reactions of the oxidase and reductases ofParacoccus denitrificans with cytochromesc

Electron transport in theParacoccus denitrificans respiratory chain system is considerably more rapid when it includes the membrane-bound cytochromec ₅₅₂ than with either solubleParacoccus c ₅₅₀ or bovine cytochromec; a pool function for cytochromec is not necessary. Low concentrations ofParacoccus or bovine cytochromec stimulate the oxidase activity. This observation could explain the multiphasic Scatchard plots which are obtained. A negatively charged area on the back side ofParacoccus c which is not present in mitochondrialc could be a control mechanism forParacoccus reactions.Paracoccus oxidase and reductase reactions with bovinec show the same properties as mammalian systems; and this is true ofParacoccus oxidase reactions with its own soluble cytochromec if added polycation masks the negatively charged area. Evidence for different oxidase and reductase reaction sites on cytochromec include: (1) stimulation of the oxidase but not reductase by a polycation; (2) differences in the inhibition of the oxidase and reductases by monoclonal antibodies toParacoccus cytochromec; and (3) reaction of another bacterial cytochromec withParacoccus reductases but not oxidase. Rapid electron transport occurs in cytochromec-less mutants ofParacoccus, suggesting that the reactions result from collision of diffusing complexes.     

Photothermal studies of CO photodissociation from mixed valence Escherichia coli cytochrome bo3

Here, we report the volume and enthalpy changes accompanying CO photodissociation from the mixed valence form of cytochrome bo3 oxidase from Escherichia coli. The results of photoacoustic calorimetry indicate two kinetic phases with distinct volume and enthalpy changes accompanying CO photodissociation from heme o3 and its transfer to CuB. The first phase occurring on a timescale of <50 ns is characterized by a volume decrease of -1.3+/-0.3 mL mol-1 and enthalpy change of 32+/-1.6 kcal mol-1. Subsequently, a volume increase of 2.9 mL mol-1 with an enthalpy change of -5.3+/-2.5 kcal mol-1 is observed with the lifetime of approximately 250 ns (this phase has not been detected in previous optical studies). These volume and enthalpy changes differ from the volume and enthalpy changes observed for CO dissociation from fully reduced cytochrome bo3 oxidase indicating that the heme o3/CuB active site dynamics are affected by the redox state of heme b.     

Insight into the active-site structure and function of cytochrome oxidase by analysis of site-directed mutants of bacterial cytochromeaa 3 and cytochromebo

Cytochromeaa ₃ ofRhodobacter sphaeroides and cytochromebo ofE. coli are useful models of the more complex cytochromec oxidase of eukaryotes, as demonstrated by the genetic, spectroscopic, and functional studies reviewed here. A summary of site-directed mutants of conserved residues in these two enzymes is presented and discussed in terms of a current model of the structure of the metal centers and evidence for regions of the protein likely to be involved in proton transfer. The model of ligation of the hemea ₃ (oro)-Cu_B center, in which both hemes are bound to helix X of subunit I, has important implications for the pathways and control of electron transfer.     

A reaction cycle for cytochrome c oxidase as an electron-transport-driven proton pump: The effect of electrochemical potential and slips

A detailed reaction cycle for cytochrome oxidase, an electron-transport-driven proton pump, has been presented earlier by our research group. The essential feature of the model is that both cytochrome a and Cu_A must be reduced in order to allow the transition from the electron and proton input state to the output state. The model is thus based on an indirect coupling between electron transfer and proton translocation.In this study, the same model is examined with respect to (1) intrinsic electron and proton leaks and (2) the effect of applying an electrochemical potential gradient on the pump incorporated in a membrane, both with respect to the electrical and chemical components.The model is successfully used to simulate various experimental results. Comparisons of experimental results with simulations based on the model support the existence of electron and proton leaks. The analysis of electron leaks suggests that electron gating is best achieved by varying the reorganization energy rather than by varying the reduction potentials.It is also suggested that both the electrical and chemical components of the electrochemical potential gradient are responsible for the regulation of the enzyme activity. Furthermore, an attempt is made to interpret the seemingly contradictory results obtained when measuring the pH dependence of the reduction potential of cytochrome a. In addition, the simulations support the assumption that protons are pumped by a mechanism that combines a membrane Bohr effect with the transition-state mechanism.Abbreviations R molar gas constant - k _B Boltzmann contant - F Faraday constant - e elementary charge - T absolute temperature - transmembrane electrochemical potential gradient - pH transmembrane pH difference - pH₁ and pH₂ inside (matrix) and outside (cytosol) pH, respectively - transmembrane electrical potential - E _m midpoint potential     

Ataxia telangiectasia mutated influences cytochrome c oxidase activity

Cells lacking ataxia telangiectasia mutated (ATM) have impaired mitochondrial function. Furthermore, mammalian cells lacking ATM have increased levels of reactive oxygen species (ROS) as well as mitochondrial DNA (mtDNA) deletions in the region encoding for cytochrome c oxidase (COX). We hypothesized that ATM specifically influences COX activity in skeletal muscle. COX activity was ∼40% lower in tibialis anterior from ATM-deficient mice than for wild-type mice (P < 0.01, n = 9/group). However, there were no ATM-related differences in activity of succinate dehydrogenase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, mitochondrial glycerol 3-phosphate dehydrogenase, or complex III. Incubation of wild-type extensor digitorum longus muscles for 1 h with the ATM inhibitor KU55933 caused a ∼50% reduction (P < 0.05, n = 5/group) in COX activity compared to muscles incubated with vehicle alone. Among the control muscles and muscles treated with the ATM inhibitor, COX activity was correlated (r = 0.61, P < 0.05) with activity of glucose 6-phosphate dehydrogenase, a key determinant of antioxidant defense through production of NADPH. Overall, the findings suggest that ATM has a protective role for COX activity.     

The regulation of cytochrome c oxidase of Rhodobacter capsulatus by light and oxygen

In order to distinguish between the regulatory effects of oxygen tension and light intensity on cytochrome c oxidase protein and enzymatic activity cells of Rhodobacter capsulatus were shifted from phototrophic (anaerobic, light) growth to aerobic-light, aerobic-dark and to anaerobic-dark conditions, respectively. During shift-experiments the formation of oxidase protein and regulation of oxidase activity was followed by immunological and enzymatic means. The results support the idea, that the formation of oxidase protein is regulated by oxygen tension and light intensity changes, whereas the regulation of oxidase activity seems only to be correlated to the oxygen tension. A DNA sequence involved in the oxygen-dependent regulation of cytochrome oxidase could be identified in the regulation-deficient oxidase mutant H41 of R. capsulatus. Immunological investigations of cytochrome c ₂ from mutant H41 demonstrated at the same time the participation of the c ₂-polypeptide in the regulation of cytochrome c oxidase.Abbreviations Bchl bacteriochlorophyll - CIE crossed immuno-electrophoresis - DMSO dimethyl sulfoxide     

Superoxide dismutase does not inhibit the oxidation of cytochrome c and cytochrome oxidase.

An electrometric system was used to measure Ca⁺⁺ uptake by sarcoplasmic reticulum vesicles (SR). The method permits continuous recording of Ca⁺⁺ uptake and thus the valuation of kinetic parameters. Furthermore, the ultrasensitivity of the method permits to follow changes in Ca⁺⁺ concentration below 10^?6 M.     

Interactions in cytochrome oxidase: Functions and structure

Mitochondrial cytochromec oxidase is an exceedingly complex multistructural and multifunctional membranous enzyme. In this review, we will provide an overview of the many interactions of cytochrome oxidase, stressing developments not covered by the excellent monograph of Wikström, Krab, and Saraste (1981), and continuing into early 1983. First we describe its functions (both in the nominal sense, as a transporter of electrons between cytochromec and oxygen, and in its role in energy transduction). Then we describe its structure, emphasizing the protein (its structure as a whole, the number and stoichiometry of its subunits, their biosynthetic origin, and their interactions with each other, with other components of the enzyme complex, and with the membrane as a whole). Finally, we present a model in which the protein conformation serves as the focus for the dynamic interaction of its two major functions.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - E _m midpoint potential - EPR electron paramagnetic resonance - F₁ soluble portion of the ATP synthetase complex - NMR nuclear magnetic resonance - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate - SUPAGE SDS-urea-PAGE     

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.     

A comparison of catalytic site intermediates of cytochrome <Emphasis Type="Italic">c</Emphasis> oxidase and peroxidases

Compounds I and II of peroxidases such as horseradish peroxidase and cytochrome c peroxidase are relatively well understood catalytic intermediates in terms of their structures and redox states of iron, heme, and associated radical species. The intermediates involved in the oxygen reduction chemistry of the cytochrome c oxidase superfamily are more complicated because of the need for four reducing equivalents and because of the linkage of the oxygen chemistry with vectorial proton translocations. Nevertheless, two of these intermediates, the peroxy and ferryl forms, have characteristics that can in many ways be considered to be counterparts of peroxidase compounds I and II. We explore the primary factors that minimize the generation of unwanted reactive oxygen species products and ensure that the principal enzymological function becomes either that of a peroxidase or an oxidase. These comparisons can provide insights into the nature of biological oxygen reduction chemistry and guidance for the engineering of biomimetic synthetic materials. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 10, pp. 1289–1299.     

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.     

The cytochrome composition of the meat spoilage bacterium Brochothrix thermosphacta: identification of cytochrome a 3- and d-type terminal oxidases under various conditions

Brochothrix thermosphacta, grown in batch culture in a yeast-dextrose broth, at temperatures from 30 °C to 10 °C, contained diverse membrane-bound respiratory cytochromes. Under conditions of moderate aeration, cytochromes of the a-, b- and d-type were detected at all growth temperatures, but the proportions changed as a function of temperature, with the spectra of cells grown at 10 or 15 °C being dominated by a-type cytochrome(s). Cytochrome a ₃ was detected by its reactions with CO and cyanide in cells from all growth conditions. An additional cytochrome a, which was not cyanide-reactive, was also detected, suggesting the presence of an aa ₃ oxidase complex. Cytochrome d was cyanide- and CO-reactive, but not detectable in photodissociation spectra, presumably because of the very rapid recombination of CO at the sub-zero temperatures used. Decreasing the oxygen transfer rates to batch cultures resulted in enhanced expression of cytochrome d and changed the proportion of the aa ₃-type oxidase that could be attributed to ligand-binding cytochrome a ₃; at the lowest oxygen transfer rates, no cytochrome a was detected, suggesting the presence of a cytochrome ba ₃ terminal oxidase complex. Intact cells showed no evidence of a c-type cytochrome and no haem C was detected in membrane preparations. After growth at 10°C, the cytochrome composition of B. campestris was essentially identical to that of B. thermosphacta. The multiplicity of putative terminal oxidases in B. thermosphacta is discussed.     

Redox-coupled proton pumping in cytochrome c oxidase: Further insights from computer simulation

The membrane-bound enzyme cytochrome c oxidase, the terminal member in the respiratory chain, converts oxygen into water and generates an electrochemical gradient by coupling the electron transfer to proton pumping across the membrane. Here we have investigated the dynamics of an excess proton and the surrounding protein environment near the active sites. The multi-state empirical valence bond (MS-EVB) molecular dynamics method was used to simulate the explicit dynamics of proton transfer through the critically important hydrophobic channel between Glu242 (bovine notation) and the D-propionate of heme a₃ (PRDa₃) for the first time. The results from these molecular dynamics simulations indicate that the PRDa₃ can indeed re-orientate and dissociate from Arg438, despite the high stability of such an ion pair, and has the ability to accept protons via bound water molecules. Any large conformational change of the adjacent heme a D-propionate group is, however, sterically blocked directly by the protein. Free energy calculations of the PRDa₃ side chain isomerization and the proton translocation between Glu242 and the PRDa₃ site have also been performed. The results exhibit a redox state-dependent dynamical behavior and indicate that reduction of the low-spin heme a may initiate internal transfer of the pumped proton from Glu242 to the PRDa₃ site.