Electronic and vibrational spectroscopy of the cytochrome c:cytochrome c oxidase complexes from bovine and Paracoccus denitrificans.

The 1:1 complex between horse heart cytochrome c and bovine cytochrome c oxidase, and between yeast cytochrome c and Paracoccus denitrificans cytochrome c oxidase have been studied by a combination of second derivative absorption, circular dichroism (CD), and resonance Raman spectroscopy. The second derivative absorption and CD spectra reveal changes in the electronic transitions of cytochrome a upon complex formation. These results could reflect changes in ground state heme structure or changes in the protein environment surrounding the chromophore that affect either the ground or excited electronic states. The resonance Raman spectrum, on the other hand, reflects the heme structure in the ground electronic state only and shows no significant difference between cytochrome a vibrations in the complex or free enzyme. The only major difference between the Raman spectra of the free enzyme and complex is a broadening of the cytochrome a3 formyl band of the complex that is relieved upon complex dissociation at high ionic strength. These data suggest that the differences observed in the second derivative and CD spectra are the result of changes in the protein environment around cytochrome a that affect the electronic excited state. By analogy to other protein-chromophore systems, we suggest that the energy of the Soret pi* state of cytochrome a may be affected by (1) changes in the local dielectric, possibly brought about by movement of a charged amino acid side chain in proximity to the heme group, or (2) pi-pi interactions between the heme and aromatic amino acid residues.     

A pivotal role for p53: balancing aerobic respiration and glycolysis

The genetic basis of increased glycolytic activity observed in cancer cells is likely to be the result of complex interactions of multiple regulatory pathways. Here we review the recent evidence of a simple genetic mechanism by which tumor suppressor p53 regulates mitochondrial respiration with secondary changes in glycolysis that are reminiscent of the Warburg effect. The biological significance of this regulation of the two major pathways of energy generation by p53 remains to be seen.     

A Gene Encoding a Cytochrome c Oxidase-Like Protein Is Located Closely to the Cytochrome c-553 Gene in the Anaerobic Bacterium,Desulfovibrio vulgaris (Miyazaki F)

The gene encoding cytochrome c-553 from Desulfovibrio vulgaris (Miyazaki F) was cloned using a synthetic oligodeoxyribonucleotide probe. The nucleotide sequence indicated that cytochrome c-553 was synthesized as a precursor protein with an NH₂-terminal signal sequence of 23 residues. In the cloned DNA fragment, there are three other open reading frames whose products have 191, 157, 541 amino acid residues, respectively. The putative ORF-4 product is highly homologous with the cytochrome c oxidase subunit I from various organisms.     

Methods for assaying cyanide in bacterial culture supernatant

AIMS: To find an easy, rapid and direct method for the quantitation of cyanide in a moderate number of bacterial culture supernatants. METHODS AND RESULTS: Culture supernatant from stationary phase cultures of Pseudomonas aeruginosa, grown in LB media, were analysed for cyanide content using the Merckoquant and Spectroquant cyanide detection kits as well as a cyanide ion-selective electrode (ISE) and a cyanide micro-ISE. The Merckoquant kit, designed for detection of low quantities of cyanide in water systems, proved not to be sufficiently reliable, providing poor comparison with previous assessments of cyanide levels in Ps. aeruginosa. The Spectroquant kit, and the two ISEs all provided very similar results, in agreement with previous data; however, it was the ISEs that fulfilled all the criteria for a rapid, direct test in a moderate number of samples. CONCLUSIONS: Cyanide ISEs can be used for easy assessment of the cyanide quantity in cultures grown in LB medium. Significance and Impact of the Study: The use of a cyanide ISE allows for an easy, direct and reproducible method for assaying cyanide in bacterial culture supernatant, which is of significant advantage over the currently accepted methods. This is especially important in an era of high-output genomic studies for assessing the phenotypic significance of data relating to the cyanide synthetic genes.     

Oxygen and proton pathways in cytochrome c oxidase

Cytochrome c oxidase is a redox-driven proton pump, which couples the reduction of oxygen to water to the translocation of protons across the membrane. The recently solved x-ray structures of cytochrome c oxidase permit molecular dynamics simulations of the underlying transport processes. To eventually establish the proton pump mechanism, we investigate the transport of the substrates, oxygen and protons, through the enzyme. Molecular dynamics simulations of oxygen diffusion through the protein reveal a well-defined pathway to the oxygen-binding site starting at a hydrophobic cavity near the membrane-exposed surface of subunit I, close to the interface to subunit III. A large number of water sites are predicted within the protein, which could play an essential role for the transfer of protons in cytochrome c oxidase. The water molecules form two channels along which protons can enter from the cytoplasmic (matrix) side of the protein and reach the binuclear center. A possible pumping mechanism is proposed that involves a shuttling motion of a glutamic acid side chain, which could then transfer a proton to a propionate group of heme α₃. Proteins 30:100–107, 1998. © 1998 Wiley-Liss, Inc.     

Does the low respiratory rate in unfertilized eggs result mainly from depression of the redox reaction catalyzed by flavoproteins? Analysis of the respiratory system by light-induced release of CO-mediated inhibition

In unfertilized eggs of the sea urchin, the quite low respiratory rate is enhanced by tetramethyl- p -phenylenediamine (TMPD), phenazine methosulfate (PMS) and sperm and this augmentation is completely inhibited by carbon monoxide (CO). Exposure to light releases eggs from this CO-mediated inhibition. The action spectra for photoreactivation of CO-inhibited cytochrome c oxidase in isolated mitochondria and CO-blocked respiration in TMPD-treated eggs were found to be similar to the absorption spectrum of CO-bound cytochrome aa ₃. In PMS-treated eggs and fertilized eggs, the maximum photoreactivation of CO-inhibited respiration occurred at a light fluence rate higher than that for maximum photoreactivation of CO-inhibited respiration in TMPD-treated eggs, with peaks at the same wavelengths as those in the absorption spectrum of reduced cytochrome b. A similar phenomenon was seen for NADH cytochrome c reductase in mitochondria. Thus, cytochrome c oxidase and NADH cytochrome c reductase, whose activities are not altered by fertilization, seem to be functional, even in unfertilized eggs. In unfertilized eggs, difference spectra indicated that PMS and sperm augmented cytochrome b reduction and that TMPD accelerated cytochrome c reduction without cytochrome b reduction. Therefore, it is likely that depression of electron transport to cytochrome b , which is augmented by PMS and sperm, is responsible for the low respiratory rate in unfertilized eggs.     

Tracing hybrid incompatibilities to single amino acid substitutions

Deleterious interactions among genes cause reductions in fitness of interpopulation hybrids (hybrid breakdown). Identifying genes involved in hybrid breakdown has proven difficult, and few studies have addressed the molecular basis of this widespread phenomenon. Because proper function of the mitochondrial electron transport system (ETS) requires a coadapted set of nuclear and mitochondrial gene products, ETS genes present an attractive system for studying the evolution of coadapted gene complexes within isolated populations and the loss of fitness in interpopulation hybrids. Here we show the effects of single amino acid substitutions in cytochrome c (CYC) on its functional interaction with another ETS protein, cytochrome c oxidase (COX) in the intertidal copepod Tigriopus californicus. The individual and pairwise consequences of three naturally occurring amino acid substitutions in CYC are examined by site-directed mutagenesis and found to differentially effect the rates of CYC oxidation by COX variants from different source populations. In one case, we show that interpopulation hybrid breakdown in COX activity can be attributed to a single naturally occurring amino acid substitution in CYC.     

Pb-induced alterations in tyrosine hydroxylase activity in rat brain

It was concluded that cytochrome oxidase was a strange enzyme for three reasons. (1) The thermodynamic flux-force relationship of this enzyme was inverse in some conditions: flux decreased when force increased. (2) The flux-force relationship was not unique and depended on the way in which the thermodynamic span of cytochrome oxidase was changed. (3) The regulation of cytochrome oxidase was different in the same conditions when different external parameters (energy demand, oxygen concentration) were changed.It was also shown that the flux control coefficient of cytochrome oxidase, small at saturating oxygen concentration, increases when oxygen pressure diminishes, approaching unity at very low oxygen concentrations. (Mol Cell Biochem 174: 137–141, 1997)     

Incongruence of morphology and genetic markers in Hyles tithymali (Lepidoptera: Sphingidae) from the Canary Islands

Hyles t. tithymali on the Canary Islands has been observed to occur in two larval morphotypes, connected by intermediate forms along a geographical cline from east to west. In this study, it was tested whether this distribution of phenotypes reflects a genealogical division of the population. mtDNA sequence data (COI + II, tRNA-leu) and genomic fingerprints from intersimple sequence repeat (ISSR)-PCR data were used. The sequence data had low variation (max. 0.4%), and phylogenetic analyses did not reveal groups that correlated with the morphotype. The samples did not group according to their island of origin and the most common haplotype was shared among all islands. Although nine haplotypes occurred only on the westernmost islands, the data showed little phylogeographical structure. The population of H. t. tithymali appears to reflect a comparatively rapid and recent colonization event of the Canary Islands. The ISSR-PCR data were very variable and did not reveal patterns corresponding to morphological variation or geographical distribution. Although the two morphs observed may represent the first stage of differentiation between two lineages, the recent origin of H. t. tithymali provided insufficient time for complete lineage sorting of ancestral polymorphism. Hence, the population of Hyles t. tithymali on the Canary Islands appears genetically more homogeneous than that was expected from the phenotypic distribution of the two morphotypes in the population.     

Probing protein-cofactor interactions in the terminal oxidases by second derivative spectroscopy: study of bacterial enzymes with cofactor substitutions and heme A model compounds.

Second derivative absorption spectra are reported for the aa3-cytochrome c oxidase from bovine cardiac mitochondria, the aa3-600 ubiquinol oxidase from Bacillus subtilis, the ba3-cytochrome c oxidase from Thermus thermophilis, and the aco-cytochrome c oxidase from Bacillus YN-2000. Together these enzymes provide a range of cofactor combinations that allow us to unequivocally identify the origin of the 450-nm absorption band of the terminal oxidases as the 6-coordinate low-spin heme, cytochrome a. The spectrum of the aco-cytochrome c oxidase further establishes that the split Soret band of cytochrome a, with features at 443 and 450 nm, is common to all forms of the enzyme containing ferrocytochrome a and does not depend on ligand occupancy at the other heme cofactor as previously suggested. To test the universality of this Soret band splitting for 6-coordinate low-spin heme A systems, we have reconstituted purified heme A with the apo forms of the heme binding proteins, hemopexin, histidine-proline-rich glycoprotein and the H64V/V68H double mutant of human myoglobin. All 3 proteins bound the heme A as a (bis)histidine complex, as judged by optical and resonance Raman spectroscopy. In the ferroheme A forms, none of these proteins displayed evidence of Soret band splitting. Heme A-(bis)imidazole in aqueous detergent solution likewise failed to display Soret band splitting. When the cyanide-inhibited mixed-valence form of the bovine enzyme was partially denatured by chemical or thermal means, the split Soret transition of cytochrome a collapsed into a single band at 443 nm.(ABSTRACT TRUNCATED AT 250 WORDS)