The water channel of cytochrome c oxidase: inferences from inhibitor studies

Cytochrome c oxidase couples electron transfer to proton transfer from inside the mitochondrion to the cytosol. Protons pass through a channel; it is closed except when protons are pumped. Electron transfer is also coupled to a water cycle. Water moves into and out of the oxidase during electron transfer, presumably through a channel. The three processes are coupled because of the common dependence on electron transfer. If water and protons had to pass through the same channel for the proton to pass, it might be possible to block the pore by entraining small molecules in the flow. The data in this report indicate that there is a correlation between the ability of a compound to inhibit the oxidase and its size. Formamide and formaldehyde are potent inhibitors. Larger and smaller molecules are poor inhibitors. Formamide introduces an internal block in electron transfer. It is a slow-onset, reversible inhibitor, dependent on turnover to manifest its effects. Vesicular oxidase is less influenced by formamide than is soluble oxidase; formamide must pass a permeability barrier to act. The data are consistent with a proton channel with constrictions at both ends that open to yield a pore of approximately 4 A.     

The relationship between evolutionary theory and phylogenetic analysis

The relationship between phylogenetic reconstruction and evolutionary theory is reassessed. It is argued here that phylogenies, and evolutionary principles, should be analysed initially as independently from each other as possible. Only then can they be used to test one another. If the phylogenies and evolutionary principles are totally consistent with one another, this consilience of independent lines of evidence increases confidence in both. If, however, there is a conflict, then one should assess the relative support for each hypothesis, and tentatively accept the more strongly supported one. We review examples where the phylogenetic hypothesis is preferred over the evolutionary principle, and vice versa, and instances where the conflict cannot be readily resolved. Because the analyses of pattern and process must initially be kept separate, the temporal order in which they are performed is unimportant. Therefore, the widespread methodology of always proceeding from cladogram to evolutionary 'scenario' cannot be justified philosophically. Such an approach means that cladograms cannot be properly tested against evolutionary principles, and that evolutionary 'scenarios' have no independent standing. Instead, we propose the 'consilience' approach where phylogenetic and evolutionary hypotheses are formulated independently from each other and then examined for agreement.     

Molecular evolutionary dynamics of cytochrome b in strepsirrhine primates: the phylogenetic significance of third-position transversions

DNA sequences of the complete cytochrome b gene are shown to contain robust phylogenetic signal for the strepsirrhine primates (i.e., lemurs and lorises). The phylogeny derived from these data conforms to other molecular studies of strepsirrhine relationships despite the fact that uncorrected nucleotide distances are high for nearly all intrastrepsirrhine comparisons, with most in the 15%-20% range. Cytochrome b sequences support the hypothesis that Malagasy lemuriforms and Afro-Asian lorisiforms each comprise clades that share a sister-group relationship. A study (Adkins and Honeycutt 1994) of the cytochrome c oxidase subunit II (COII) gene placed one Malagasy primate (Daubentonia) at the base of the strepsirrhine clade, thereby suggesting a diphyletic Lemuriformes. The reanalysis of COII third-position transversions, either alone or in combination with cytochrome b third-position transversions, however, yields a tree that is congruent with phylogenetic hypotheses derived from cytochrome b and other genetic data sets.     

Adenylosuccinate synthetase genes: molecular cloning and phylogenetic analysis of a highly conserved archaeal gene

Adenylosuccinate synthetase (PurA) catalyzes the first step in the de novo AMP synthesis and has been extensively studied in both Bacteria and Eukarya. We cloned the purA gene from the hyperthermophilic archaeon, Pyrococcus furiosus. The gene appears to be individually transcribed and encodes a protein of 339 amino acids. The amino acid sequence comparison with other archael PurAs found from recent genome analyses indicated that two deletions, one central and the other C-terminal, are a common feature of archaeal PurAs. None of the 21 PurA homologues analyzed from Eukarya and Bacteria exhibited this feature. Amino acid sequences of PurAs in Archaea showed 64% average identities which were significantly higher than the 50% and 55% calculated for Bacteria and Eukarya, respectively. Several residues conserved in PurAs of both Eukarya and Bacteria and shown to be of catalytic importance are missing in the archaeal PurAs. Phylogenetic analysis using PurA as the marker grouped life into 3 domains, hence it was consistent with results derived from 16-18S ribosomal RNA sequences. The topology within the three domains, in general, portrayed the hitherto accepted evolutionary relationship among the organisms utilized. PurA can, thus, serve as an additional marker to evaluate phylogenetic inferences drawn from sequence data from rRNA and other conserved genes. The presence of two unique deletions in both euryarchaeal and crenarchaeal PurAs, but not in those of Bacteria and Eukarya, is a strong evidence confirming the common lineage of these two subdomains of Archaea.     

Laminar and columnar patterns of geniculocortical projections in the cat: relationship to cytochrome oxidase

We examined the laminar and columnar arrangement of projections from different layers of the lateral geniculate nucleus (LGN) to the visual cortex in the cat. In light of recent reports that cytochrome oxidase blobs (which in primates receive specific geniculate inputs) are also found in the visual cortex of cats, the relationship between cytochrome oxidase staining and geniculate inputs in this species was studied. Injections of wheat germ agglutinin-conjugated horseradish peroxidase were made into the anterior "genu" of the LGN, where isoelevation contours of the geniculate layers are distorted due to the curvature of the nucleus. Consequently, anterograde labeling from the various LGN layers was topographically separated across the surface of the cortex, and labeling in a particular isoelevation representation of the cortex could be associated with a specific layer of the LGN. Labeling from the A layers, which contain X and Y cells, was coextensive with layers 4 and 6 in both area 17 and area 18, as previously reported. Labeling from the C layers, which contain Y and W cells, occupied a zone extending from the 4a/4b border to part way into layer 3 in area 17. The labeling extended throughout layer 4 in area 18. There was also labeling in layer 5a and layer 1 in both area 17 and area 18. Except in layer 1, labeling from the C layers was patchy. In the tangential plane, adjacent sections stained for cytochrome oxidase showed that the patches of labeling from the C laminae aligned with the cytochrome oxidase blobs. The cytochrome blobs were visible in layers 3 and 4a, but not in layer 4b in both areas 17 and 18. These results suggest that W cells project specifically to the layer 3 portion of the blobs, while Y cells, at least those of the C layers, project specifically to the layer 4a portion of the blobs in area 17. The heavy synaptic drive of the Y cells is probably the cause of the elevated metabolism, and thus, higher cytochrome oxidase activity, of the blobs.     

Thermodynamic volume cycles for electron transfer in the cytochrome c oxidase and for the binding of cytochrome c to cytochrome c oxidase

Determining the way in which deleterious mutations interact in their effects on fitness is crucial to numerous areas in population genetics and evolutionary biology. For example, if each additional mutation leads to a greater decrease in log fitness than the last (synergistic epistasis), then the evolution of sex and recombination may be favored to facilitate the elimination of deleterious mutations. However, there is a severe shortage of relevant data. Three relatively simple experimental methods to test for epistasis between deleterious mutations in haploid species have recently been proposed. These methods involve crossing individuals and examining the mean and/or skew in log fitness of the offspring and parents. The main aim of this paper is to formalize these methods, and determine the most effective way in which tests for epistasis could be carried out. We show that only one of these methods is likely to give useful results: crossing individuals that have very different numbers of deleterious mutations, and comparing the mean log fitness of the parents with that of their offspring. We also reconsider experimental data collected on Chlamydomonas moewussi using two of the three methods. Finally, we suggest how the test could be applied to diploid species.     

A metabolic map of cytochrome oxidase in the rat brain: histochemical, densitometric and biochemical studies

To examine brain patterns of metabolic and functional activity, the distribution of cytochrome oxidase, a mitochondrial enzyme marker for neuronal functional activity, was mapped throughout the rat brain. Mapping was done qualitatively by enzyme histochemistry of brain sections cut in three planes (coronal, sagittal and horizontal), and quantitatively by optical densitometry of stained sections and by biochemical assays of brain tissue homogenates. Activity of the enzyme was distributed in characteristic patterns and amounts that differed among various neural pathways, brain nuclei, cerebral cortical areas and layers, and neuron types. Gray matter essentially always had higher enzyme activity than did white matter, by a factor of eight- to 12-fold. Among different neural pathways, cytochrome oxidase activity was relatively high in special sensory, somatosensory and motor systems, and was relatively low in associative, limbic, autonomic and visceral regulatory systems (though exceptional areas were present). Among 11 different neuron types, nearly a two-fold range of histochemical staining intensities was observed, with the darkest staining in neurons of the mesencephalic trigeminal nucleus. The observed patterns of cytochrome oxidase activity were mostly similar to the patterns of 2-deoxyglucose uptake seen previously [Schwartz W. J. and Sharp F. R. (1978) J. comp. Neurol. 177, 335-360; Sokoloff L. et al. (1977) J. Neurochem. 28, 897-916] in conscious, "resting" animals, though some differences were found. For example, whereas 2-deoxyglucose uptake was about three-fold higher in gray matter than in white matter [Sokoloff L. et al. (1977) J. Neurochem. 28, 897-916], cytochrome oxidase activity was about eight- to 12-fold higher. This and other discrepancies probably reflect basic technical differences between these two methods. Compared to 2-deoxyglucose, cytochrome oxidase is more specific for oxidative rather than glycolytic metabolism, and more reflective of overall neuronal functional activity occurring over longer time periods lasting hours to weeks, rather than minutes. The anatomical resolution of cytochrome oxidase histochemistry is also finer than that of 2-deoxyglucose autoradiography, extending to the electron microscopic level. The metabolic map of cytochrome oxidase activity reveals patterns of normal brain function, and may be useful as a baseline for comparison in studies of brain disease, development, ageing and plasticity.     

Phylogenetic affinity of mitochondria of Euglena gracilis and kinetoplastids using cytochrome oxidase I and hsp60

To elucidate the role of ras gene mutations during the early stage of colorectal tumour progression, K-ras gene mutations were analysed in 32 benign adenomas and 36 adenomas with focal carcinoma in the colorectum by microscraping of histologically pure regions from tissue sections, polymerase chain reaction-restriction fragment length polymorphism and in part by direct sequencing. Several regions were scraped out and analysed when an adenoma contained areas with different grades of dysplasia. The frequencies of K-ras gene mutation in mild dysplasia, moderate dysplasia and focal carcinoma were 19% (7/36), 51% (25/49) and 39% (14/36) respectively. The K-ras gene status was heterogeneous in 4 of the 11 benign adenomas from which multiple samples were obtained, and mutations were always found in the regions with more advanced dysplasia in these adenomas. Thirteen of the 36 adenomas with focal carcinoma showed heterogeneity of mutations between the adenoma region and the focal carcinoma. Seven of which had mutations only in the adenoma region. These findings indicated that the K-ras gene mutations occur during the late stage of adenoma progression and may confer a more advanced morphological phenotype of adenoma, but these mutations are not mainly involved in malignant transformation from adenoma to carcinoma.     

Structural and evolutionary studies on sterol 14-demethylase P450 (CYP51), the most conserved P450 monooxygenase: II. Evolutionary analysis of protein and gene structures

Phylogenetic analyses based on protein sequence data indicated that sterol 14-demethylase P450 (CYP51) and bacterial CYP51-like protein were joined into a distinctive evolutionary cluster, CYP51 cluster, within the CYP protein superfamily. The most probable branch topology of the CYP51 phylogenetic tree was (bacteria, (plants, (fungi, mammals))), which is comparable to the phylogeny of major kingdoms of living matter, suggesting that CYP51 has been conserved from the era of prokaryotic evolution. This may be strong evidence supporting the prokaryotic origin of P450. Structure of flanking regions and the number and insertion sites of introns are quite different between mammalian and fungal CYP51s. This fact indicates that different mechanisms are operative in evolution of protein sequences and gene structures. CYP51 is the first example violating the well-documented rule that the basic structure of a gene, including intron insertion sites, is well conserved in each P450 family. One CYP51 processed a pseudogene was found in rat genome. Nonsynonymous nucleotide divergence observed between the pseudogene and CYP51 cDNA was less than one-fifth of the synonymous divergence. This unusually low rate of nonsynonymous nucleotide changes in the pseudogene suggests that it may be derived from another CYP51, which might have been active for a significant duration in the past.     

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.     

Sequence analysis of three deficient mutants of cytochrome oxidase subunit I of Saccharomyces cerevisiae and their revertants

Three respiratory-deficient mutants of cytochrome oxidase subunit I in the yeast mitochondrion have been sequenced. They are located in, or near, transmembrane segment VI, the catalytic core of the enzyme. Respiratory-competent revertants have been selected and studied. The mutant V244M was found to revert at the same site in valine (wild-type), isoleucine or threonine. The revertants of the mutant G251R were of three types: glycine (wild-type), serine and threonine at position 251. A search for second-site mutations was carried out but none were found. Among 60 revertants tested, the mutant K265M was found to revert only to the wild-type allele.     

Characterization of COX17, a yeast gene involved in copper metabolism and assembly of cytochrome oxidase

Mutations in the COX17 gene of Saccharomyces cerevisiae cause a respiratory deficiency due to a block in the production of a functional cytochrome oxidase complex. Because cox17 mutants are able to express both the mitochondrially and nuclearly encoded subunits of cytochrome oxidase, the Cox17p most likely affects some late posttranslational step of the assembly pathway. A fragment of yeast nuclear DNA capable of complementing the mutation has been cloned by transformation of the cox17 mutant with a library of genomic DNA. Subcloning and sequencing of the COX17 gene revealed that it codes for a cysteine-rich protein with a molecular weight of 8,057. Unlike other previously described accessory factors involved in cytochrome oxidase assembly, all of which are components of mitochondria, Cox17p is a cytoplasmic protein. The cytoplasmic location of Cox17p suggested that it might have a function in delivery of a prosthetic group to the holoenzyme. A requirement of Cox17p in providing the copper prosthetic group of cytochrome oxidase is supported by the finding that a cox17 null mutant is rescued by the addition of copper to the growth medium. Evidence is presented indicating that Cox17p is not involved in general copper metabolism in yeast but rather has a more specific function in the delivery of copper to mitochondria.     

Light-induced structural changes in cytochrome c oxidase: implication for the mechanism of electron and proton gating

We have investigated electrogenic events and absorbance changes following pulsed illumination of partly reduced cytochrome c oxidase in the absence of dioxygen and carbon monoxide (Hallén et al. (1993) FEBS Lett. 318, 134-138). In both types of experiment similar kinetics were observed; a rapid (tau < 0.5 micros) change was followed by relaxations with time constants of approx. 7 micros and 80 micros. Both the time constant and the activation energy of the 80 micros component were, within the experimental error, the same as those of one of the steps in the reduction of dioxygen by reduced cytochrome c oxidase. The absorbance changes showed a rapid haem reduction, followed by reoxidation. They were affected by CN(-) and N(-)3, ligands which bind in the binuclear centre of cytochrome c oxidase; the absorbance changes were quenched by CN(-) and in the presence of N(-)3, the amplitude of the 7 micros component increased whereas that of the 80 micros decreased. Based on these findings, a model is proposed which involves electron transfer from Cu(+)B to Fe(3+)A3, as a response to structural changes upon pulsed illumination. The same structural changes are also suggested to take place in the oxygen reduction. These changes may play an important role in the gating of electrons as well as protons, an obligatory feature of a redox-linked proton pump.     

An archaeal aerotaxis transducer combines subunit I core structures of eukaryotic cytochrome c oxidase and eubacterial methyl-accepting chemotaxis proteins

Signal transduction in the archaeon Halobacterium salinarum is mediated by three distinct subfamilies of transducer proteins. Here we report the complete htrVIII gene sequence and present analysis of the encoded primary structure and its functional features. HtrVIII is a 642-amino-acid protein and belongs to halobacterial transducer subfamily B. At the N terminus, the protein contains six transmembrane segments that exhibit homology to the heme-binding sites of the eukaryotic cytochrome c oxidase. The C-terminal domain has high homology with the eubacterial methyl-accepting chemotaxis protein. The HtrVIII protein mediates aerotaxis: a strain with a deletion of the htrVIII gene loses aerotaxis, while an overproducing strain exhibits stronger aerotaxis. We also demonstrate that HtrVIII is a methyl-accepting protein and demethylates during the aerotaxis response.