Submolecular unfolding units of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> cytochrome <Emphasis Type="Italic">c</Emphasis>-551

Hydrogen exchange rates for backbone amide protons of oxidized Pseudomonas aeruginosa cytochrome c-551 (P. aeruginosa cytochrome c) have been measured in the presence of low concentrations of the denaturant guanidine hydrochloride. Analysis of the data has allowed identification of submolecular unfolding units known as foldons. The highest-energy foldon bears similarity to the proposed folding intermediate for P. aeruginosa cytochrome c. Parallels are seen to the foldons of the structurally homologous horse cytochrome c, although the heme axial methionine-bearing loop has greater local stability in P. aeruginosa cytochrome c, in accord with previous folding studies. Regions of low local stability are observed to correspond with regions that interact with redox partners, providing a link between foldon properties and function. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effect of calcium ions on electron transfer between hemes <Emphasis Type="Italic">a</Emphasis> and <Emphasis Type="Italic">a</Emphasis><Subscript>3</Subscript> in cytochrome <Emphasis Type="Italic">c</Emphasis> oxidase

Kinetics of the reduction of the hemes in cytochrome c oxidase in the presence of high concentration of ruthenium(III)hexaammine chloride was examined using a stopped-flow spectrophotometer. Upon mixing of the oxidized enzyme with dithionite and Ru(NH₃) ₆ ³⁺ , three well-resolved phases were observed: heme a reduction reaching completion within a few milliseconds is followed by two slow phases of heme a ₃ reduction. The difference spectrum of heme a ₃ reduction in the visible region is characterized by a maximum at ～612 nm, rather than at 603 nm as was believed earlier. It is shown that in the case of bovine heart cytochrome c oxidase containing a special cation-binding site in which reversible binding of calcium ion occurs, heme a ₃ reduction is slowed down by low concentrations of Ca²⁺. The effect is absent in the case of the bacterial cytochrome oxidase in which the cation-binding site contains a tightly bound Ca²⁺ ion. The data corroborate the inhibition of the cytochrome oxidase enzymatic activity by Ca²⁺ ions discovered earlier and indicate that the cation affects intramolecular electron transfer.     

Evolutionary alkaline transition in human cytochrome <Emphasis Type="Italic">c</Emphasis>

Conformational transitions in cytochrome c (cyt c) are being realized to be responsible for its multi-functions. Among a number of conformational transitions in cyt c, the alkaline transition has attracted much attention. The cDNA of human cyt c is cloned by RT-PCR and a high-effective expression system for human cyt c has been developed in this study. The equilibrium and kinetics of the alkaline transition of human cyt c have been systematically investigated for the first time, and compared with those of yeast and horse cyt c from an evolutionary perspective. The pK_a value for the alkaline transition of human cyt c is apparently higher than that of yeast and horse. Kinetic studies suggest that it is increasingly difficult for the alkaline transition of cyt c from yeast, horse and human. Molecular modeling of human cyt c shows that the omega loop where the lysine residue is located apparently further away from heme in human cyt c than in yeast iso-1 and horse heart cyt c. These results regarding alkaline conformational transition provide valuable information for understanding the molecular basis for the biological multi-functions of cyt c.     

Mitochondrial DNA diversity of <Emphasis Type="Italic">Cleruchoides noackae</Emphasis> (Hymenoptera: Mymaridae): a potential biological control agent for <Emphasis Type="Italic">Thaumastocoris peregrinus</Emphasis> (Hemiptera: Thaumastocoridae)

Thaumastocoris peregrinus Carpintero and Dellapé (Hemiptera: Thaumastocoridae) is a native Australian Eucalyptus sap-feeding insect that has become invasive and seriously damaging to commercially grown Eucalyptus in the Southern Hemisphere. Cleruchoides noackae Lin and Huber (Hymenoptera: Mymaridae) was recently discovered as an egg parasitoid of the Thaumastocoridae in Australia. Mitochondrial DNA (mtDNA; cytochrome c oxidase subunit I, COI) sequence diversity amongst 104 individuals from these native C. noackae populations revealed 24 sequence haplotypes. The COI haplotypes of individuals collected from the Sydney and Southeast Queensland clustered in distinct groups, indicating limited spread of the insect between the regions. Individuals collected from Perth in Western Australia were represented by four COI haplotypes. Although this population is geographically more isolated from other populations, two COI haplotypes were identical to haplotypes found in the Sydney region. The results suggest that C. noackae has recently been introduced into Perth, possibly from the Sydney area. The high mtDNA diversity and limited spread that is suggested for C. noackae is in contrast to the lack of geographic associated mtDNA diversity and extensive spread of T. peregrinus. If implemented as a biological control agent, this factor will need to be considered in collecting and releasing C. noackae.     

The role of individual lysine residues of horse cytochrome <Emphasis Type="Italic">c</Emphasis> in the formation of reactive complexes with components of the respiratory chain

A number of mutant forms of horse cytochrome c with single or double substitutions of lysine residues near the heme cavity involved in interaction of mitochondrial cytochrome c with ubiquinol:cytochrome c reductase (EC 1.10.2.2) (complex III) and cytochrome c oxidase (EC 1.9.3.1) (complex IV) were prepared.. The succinate:cytochrome c reductase and cytochrome c oxidase activities of mitoplasts of rat liver were measured in the presence of mutant forms of cytochrome c. The lysine residues in positions 8, 27, 72, 86, and 87 were shown to be the main contribution to the formation of a reactive complex with ubiquinol:cytochrome c reductase of the respiratory chain, whereas the lysine residues in positions 13, 79, 86, and 87 were predominantly responsible for the formation of a complex with cytochrome c oxidase.     

Identification and characterization of a heme periplasmic-binding protein in <Emphasis Type="Italic">Haemophilus ducreyi</Emphasis>

Haemophilus ducreyi, a gram-negative and heme-dependent bacterium, is the causative agent of chancroid, a genital ulcer sexually transmitted infection. Heme acquisition in H. ducreyi proceeds via a receptor mediated process in which the initial event involves binding of hemoglobin and heme to their cognate outer membrane proteins, HgbA and TdhA, respectively. Following this specific interaction, the fate of the periplasmic deposited heme is unclear. Using protein expression profiling of the H. ducreyi periplasmic proteome, a periplasmic-binding protein, termed hHbp, was identified whose expression was enhanced under heme-limited conditions. The gene encoding this protein was situated in a locus displaying genetic characteristics of an ABC transporter. The purified protein bound heme in a dose-dependent and saturable manner and this binding was specifically competitively inhibited by heme. The hhbp gene functionally complemented an Escherichia coli heme uptake mutant. Expression of the heme periplasmic-binding protein was detected in a limited survey of H. ducreyi and H. influenzae clinical strains. These results indicate that the passage of heme into the cytoplasm of H. ducreyi involves a heme dedicated ABC transporter.     

Another look at the interaction between mitochondrial cytochrome <Emphasis Type="Italic">c</Emphasis> and flavocytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>2</Subscript>

Yeast flavocytochrome b ₂ tranfers reducing equivalents from lactate to oxygen via cytochrome c and cytochrome c oxidase. The enzyme catalytic cycle includes FMN reduction by lactate and reoxidation by intramolecular electron transfer to heme b ₂. Each subunit of the soluble tetrameric enzyme consists of an N terminal b ₅-like heme-binding domain and a C terminal flavodehydrogenase. In the crystal structure, FMN and heme are face to face, and appear to be in a suitable orientation and at a suitable distance for exchanging electrons. But in one subunit out of two, the heme domain is disordered and invisible. This raises a central question: is this mobility required for interaction with the physiological acceptor cytochrome c, which only receives electrons from the heme and not from the FMN? The present review summarizes the results of the variety of methods used over the years that shed light on the interactions between the flavin and heme domains and between the enzyme and cytochrome c. The conclusion is that one should consider the interaction between the flavin and heme domains as a transient one, and that the cytochrome c and the flavin domain docking areas on the heme b ₂ domain must overlap at least in part. The heme domain mobility is an essential component of the flavocytochrome b ₂ functioning. In this respect, the enzyme bears similarity to a variety of redox enzyme systems, in particular those in which a cytochrome b ₅-like domain is fused to proteins carrying other redox functions.     

Peroxidase activity of cytochrome <Emphasis Type="Italic">bd</Emphasis> from <Emphasis Type="Italic">Escherichia coli</Emphasis>

Cytochrome bd from Escherichia coli is able to oxidize such substrates as guaiacol, ferrocene, benzohydroquinone, and potassium ferrocyanide through the peroxidase mechanism, while none of these donors is oxidized in the oxidase reaction (i.e. in the reaction that involves molecular oxygen as the electron acceptor). Peroxidation of guaiacol has been studied in detail. The dependence of the rate of the reaction on the concentration of the enzyme and substrates as well as the effect of various inhibitors of the oxidase reaction on the peroxidase activity have been tested. The dependence of the guaiacol-peroxidase activity on the H₂O₂ concentration is linear up to the concentration of 8 mM. At higher concentrations of H₂O₂, inactivation of the enzyme is observed. Guaiacol markedly protects the enzyme from inactivation induced by peroxide. The peroxidase activity of cytochrome bd increases with increasing guaiacol concentration, reaching saturation in the range from 0.5 to 2.5 mM, but then starts falling. Such inhibitors of the ubiquinol-oxidase activity of cytochrome bd as cyanide, pentachlorophenol, and 2-n-heptyl 4-hydroxyquinoline-N-oxide also suppress its guaiacol-peroxidase activity; in contrast, zinc ions have no influence on the enzyme-catalyzed peroxidation of guaiacol. These data suggest that guaiacol interacts with the enzyme in the center of ubiquinol binding and donates electrons into the di-heme center of oxygen reduction via heme b ₅₅₈, and H₂O₂ is reduced by heme d. Although the peroxidase activity of cytochrome bd from E. coli is low compared to peroxidases, it might be of physiological significance for the bacterium itself and plays a pathophysiological role for humans and animals.     

Peculiarities of cyanide binding to the <Emphasis Type="Italic">ba</Emphasis><Subscript>3</Subscript>-type cytochrome oxidase from the thermophilic bacterium <Emphasis Type="Italic">Thermus thermophilus</Emphasis>

Cytochrome c oxidase of the ba ₃-type from Thermus thermophilus does not interact with cyanide in the oxidized state and acquires the ability to bind heme iron ligands only upon reduction. Cyanide complexes of the reduced heme a ₃ in cytochrome ba ₃ and in mitochondrial aa ₃-type cytochrome oxidase are similar spectroscopically, but the a ₃²⁺-CN complex of cytochrome ba ₃ is strikingly tight. Experiments have shown that the K _d value of the cytochrome ba ₃ complex with cyanide in the presence of reductants of the enzyme binuclear center does not exceed 10⁻⁸ M, which is four to five orders of magnitude less than the K _d of the cyanide complex of the reduced heme a ₃ of mitochondrial cytochrome oxidase. The tightness of the cytochrome ba ₃ complex with cyanide is mainly associated with an extremely slow rate of the ligand dissociation (k _off ≤ 10⁻⁷ sec⁻¹), while the rate of binding (k _on ∼ 10² M⁻¹·sec⁻¹) is similar to the rate observed for the mitochondrial cytochrome oxidase. It is proposed that cyanide dissociation from the cytochrome ba ₃ binuclear center might be hindered sterically by the presence of the second ligand molecule in the coordination sphere of Cu_B²⁺. The rate of cyanide binding with the reduced heme a ₃ does not depend on pH in the neutral area, but it approaches linear dependence on H⁺ activity in the alkaline region. Cyanide binding appears to be controlled by protonation of an enzyme group with pK _a = 8.75.     

Cytochrome <Emphasis Type="Italic">c</Emphasis> is rapidly reduced in the cytosol after mitochondrial outer membrane permeabilization

Visible spectroscopy was used to measure real-time changes in the oxidation state of cytochrome c (cyt c) and the a-cytochromes (cyt aa₃) of cytochrome oxidase during mitochondrial outer membrane permeabilization (MOMP) initiated by anisomycin in HL-60 cells. The oxidation state of mitochondrial cyt c was found to be ≈62% oxidized before MOMP and became ≈70% oxidized after MOMP. In contrast, the cytosolic pool of cyt c was found to be almost fully reduced. This oxidation change allows cyt c release to be continuously and quantitatively monitored in real time. Anoxia and antimycin were used to fully reduce and fully oxidize, respectively, the mitochondrial pool of cyt c and it was found that the release of cyt c was independent of it oxidation state consistent with a simple model of cyt c passively diffusing down a concentration gradient through a pore or tear in the outer membrane. After MOMP was complete, the flux of cyt c diffusing back into the mitochondria was measured from the residual mitochondrial oxygen consumption after complete inhibition of the bc₁ with antimycin and myxothiazol. The outer membrane was found to be highly permeable after MOMP implying that the reduction of cyt c in the cytosol must be very rapid. The permeability of the outer membrane measured in this study would result in the release of cyt c with a time constant of less than 1 s.     

Construction and characteristics of transgenic tobacco <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> L. plants expressing <Emphasis Type="Italic">CYP11A1</Emphasis> cDNA encoding cytochrome P450<Subscript>SCC</Subscript>

In steroidogenic animal tissues cytochrome P450_SCC catalizes the conversion of cholesterol into pregnenolone, a common metabolic precursor of all steroid hormones. To study the possibility of functioning of mammalian cytochrome P450_SCC in plants and the mechanism of its integration in the plant steroidogenic system, transgenic plants of tobacco Nicotiana tabacum L. were developed carrying cDNA of CYP11A1 encoding cytochrome P450_SCC of bovine adrenal cortex. Pregnenolone, a product of the reaction catalyzed by cytochrome P450_SCC, was discovered in the steroid-containing fraction of transgenic plants. Transgenic plants are characterized by a reduced period of vegetative development (early flowering and maturation of bolls) and increased productivity. The contents of soluble protein and carbohydrates in leaves and seeds of transgenic plants are essentially higher than the contents of these components in leaves and seeds of control plants.     

Cytochrome <Emphasis Type="Italic">c</Emphasis> signalosome in mitochondria

Cytochrome c delicately tilts the balance between cell life (respiration) and cell death (apoptosis). Whereas cell life is governed by transient electron transfer interactions of cytochrome c inside the mitochondria, the cytoplasmic adducts of cytochrome c that lead to cell death are amazingly stable. Interestingly, the contacts of cytochrome c with its counterparts shift from the area surrounding the heme crevice for the redox complexes to the opposite molecule side when the electron flow is not necessary. The cytochrome c signalosome shows a higher level of regulation by post-translational modifications—nitration and phosphorylation—of the hemeprotein. Understanding protein interfaces, as well as protein modifications, would puzzle the mitochondrial cytochrome c-controlled pathways out and enable the design of novel drugs to silence the action of pro-survival and pro-apoptotic partners of cytochrome c.     

Cell-free synthesis of cytochrome <Emphasis Type="Italic">c</Emphasis> oxidase,a multicomponent membrane protein

Cell-free protein synthesis is a useful technique that can site-specifically incorporate isotope-labeled amino acids into proteins. This incorporation is essential for infrared analyses of the electronic state of a specific amino acid residue used to elucidate protein function. Although 17 membrane proteins have been synthesized in their active state by cell-free systems, to date no hetero-subunit protein has been synthesized with this technique, suggesting that there are serious technical limitations. Here we report the cell-free synthesis of Paracoccus denitrificans cytochrome c oxidase, a membrane protein complex composed of three distinct subunits that contain two heme A molecules and two redox-active copper centers. The synthesized protein exhibited normal Soret/vis absorption spectra and ferrocytochrome c oxidation activity.     

Flow-cytometry assay for apoptosis using fluorophor 10-<Emphasis Type="Italic">N</Emphasis>-nonyl acridine orange

Recently it has been shown that redox catalytic interactions of cytochrome c with cardiolipin (CL) and subsequent oxidation of CL occurs during apoptosis. Oxidation of CL is accompanied by a release of cytochrome c from the mitochondria into the cytoplasm, a key event in development of apoptosis. 10-N-Nonyl acridine orange (NAO), a fluorophor that forms a stable complex with reduced form of CL, but not with oxidized CL, can be used for flow cytometry analysis of this effect. It has been shown that after the incubation of CTLL-2 cells in the presence of 7% ethanol (90 min) and subsequent staining with NAO, a cell population with low intensity of fluorescence appears. Flow cytometry analysis of the cells by means of a conventional method (annexin V-FITC/propidium iodide (PI)) showed that the cell population with low intensity of NAO fluorescence corresponded to the population of apoptotic cells with good coincidence of percentages. Then apoptosis was induced in the cells of three lines by growth factor deprivation (IL-2-dependent cell line CTLL-2) or as a result of actinomycin D treatment, an RNA synthesis inhibitor (Jurkat and Raji cell lines). Comparison of the data obtained by a conventional assay (annexin V-FITC/PI) and a newly elaborated assay (NAO/PI) has demonstrated a good coincidence. The data obtained with these methods exhibited significant level of correlation (0.953, p < 0.0001).     

Stress tolerance of the invasive macroalgae <Emphasis Type="Italic">Codium fragile</Emphasis> and <Emphasis Type="Italic">Gracilaria vermiculophylla</Emphasis> in a soft-bottom turbid lagoon

Invasive species are often hypothesized to have superior performance traits. We compared stress tolerance (as change in biomass) of the invasive macroalgae Codium fragile ssp. tomentosoides and Gracilaria vermiculophylla to the native macroalgae Fucus vesiculosus, Agardhiella subulata, Hypnea musciformis and Ulva curvata in Hog Island Bay, a shallow lagoon in Virginia, USA. We hypothesized that the success of the two aliens is due to their high tolerances of turbidity, sedimentation, desiccation, grazing and nutrient enrichment. Like many lagoons, Hog Island Bay is characterized by extensive intertidal mudflats, high turbidity and sedimentation, and high densities of omnivorous mud snails. Nutrient enrichment may also become a problem as land use practices in adjacent watersheds change. Contrary to our hypothesis, C. fragile was less resistant to sedimentation, desiccation and grazing than other algae and had low growth at all light and nutrient levels. This suggests that any superior performance of this invasive species compared to native algae is probably limited to microhabitats where stress is minimal and where bivalve shells facilitate recruitment and long-term persistence. In contrast, G. vermiculophylla was resistant to desiccation, burial and grazing, and was not negatively influenced by either high or low light or nutrient levels. These traits reflect the current success of G. vermiculophylla in already invaded lagoons and estuaries, and indicates that it will likely continue its spread in European and North American turbid and tidal soft-sediment systems.     

The structure and function of the cytochrome <Emphasis Type="Italic">c</Emphasis><Subscript>2</Subscript>: reaction center electron transfer complex from <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

In the photosynthetic bacterium, Rhodobacter sphaeroides, the mobile electron carrier, cytochrome c₂ (cyt c₂) transfers an electron from reduced heme to the photooxidized bacteriochlorophyll dimer in the membrane bound reaction center (RC) as part of the light induced cyclic electron transfer chain. A complex between these two proteins that is active in electron transfer has been crystallized and its structure determined by X-ray diffraction. The structure of the cyt:RC complex shows the cyt c₂ (cyt c₂) positioned at the center of the periplasmic surface of the RC. The exposed heme edge from cyt c₂ is in close tunneling contact with the electron acceptor through an intervening bridging residue, Tyr L162 located on the RC surface directly above the bacteriochlorophyll dimer. The binding interface between the two proteins can be divided into two regions: a short-range interaction domain and a long-range interaction domain. The short-range domain includes residues immediately surrounding the tunneling contact region around the heme and Tyr L162 that display close intermolecular contacts optimized for electron transfer. These include a small number of hydrophobic interactions, hydrogen bonds and a pi-cation interaction. The long-range interaction domain consists of solvated complementary charged residues; positively charged residues from the cyt and negatively charged residues from the RC that provide long range electrostatic interactions that can steer the two proteins into position for rapid association.     

<Emphasis Type="Italic">Bd</Emphasis> oxidase homologue of photosynthetic purple sulfur bacterium <Emphasis Type="Italic">Allochromatium vinosum</Emphasis> is co-transcribed with a nitrogen fixation related gene

Purple sulfur bacteria, which are known to be the most ancient among anoxygenic phototrophs, play an important role in the global sulfur cycle. Allochromatium vinosum oxidizes reduced sulfur compounds such as hydrogen sulfide, elemental sulfur and thiosulfide. At low oxygen concentrations, A. vinosum can grow chemotrophically using oxygen as the terminal electron acceptor. Being also a nitrogen fixer, A. vinosum is faced with the paradox of co-existence of aerobic metabolism and nitrogen fixation. Due to growth difficulties, only a few studies have dealt with the aerobic metabolism of the organism and, until now, there has been no information about the genes involved in the respiratory metabolism of purple sulfur bacteria. In this article we show the first terminal oxidase gene for A. vinosum. The presence of a Bd type of quinol oxidase is necessary to protect nitrogenases against the inhibitory effects of oxygen. In this case, a nitrogen fixation related gene is part of the cyd operon and this gene is co-transcribed with cydAB genes. Bd oxidase of A. vinosum may be the earliest form of oxidase where the function of the enzyme is to scavenge the contaminant oxygen during nitrogen fixation. This may be an important clue about the early evolution of oxygenic photosynthesis, perhaps as a protective mechanism for nitrogen fixation.     

Electron transfer in <Emphasis Type="Italic">Paracoccus denitrificans</Emphasis> with the modified <Emphasis Type="Italic">fbc</Emphasis> operon

Membrane fragments of two mutant strains of Paracoccus denitrificans genetically modified in the bc ₁ complex have been studied for comparison of enzymic activities of succinate-cytochrome-c reductase and its components, viz. succinate dehydrogenase (Complex II) and ubiquinol-cytochrome-c reductase (Complex III) and their response to changes in concentration of succinate, cytochrome c, ionic strength, pH, temperature and sensitivity to antimycin A. The mutants synthesized and assembled the b and c hemes in the ratio characteristic for the wild type strain. The mutant strain M 71 expressing the truncated copy of cytochrome c ₁ (devoid of a stretch of 150 mainly acidic amino acids) was less sensitive to increasing concentration of cytochrome c and changes in ionic strength of the medium, but maintained the original affinity to succinate and sensitivity to antimycin A. The mutant strain M 36 with an overexpressed bc ₁ content showed the highest response to changes in ionic strength and physical parameters, exhibited the lowest turnover number values with succinate-cytochrome-c reductase, but positively affected the succinate dehydrogenase. In view of the interaction of the redox components in native membranes the functional analyses of separated Complexes II and III should be regarded with caution.     

Cloning,expression, and physicochemical characterization of a new diheme cytochrome <Emphasis Type="Italic">c</Emphasis> from <Emphasis Type="Italic">Shewanella baltica</Emphasis> OS155

The 16-kDa diheme cytochrome c from the bacterium Shewanella baltica OS155 (Sb-DHC) was cloned and expressed in Escherichia coli and investigated through UV–vis, magnetic circular dichroism, and ¹H NMR spectroscopies and protein voltammetry. The model structure was obtained by means of comparative modeling using the X-ray structure of Rhodobacter sphaeroides diheme cytochrome c (Rs-DHC) (with a 37% pairwise sequence identity) as a template. Sb-DHC folds into two distinct domains, each containing one heme center with a bis-His axial ligation. Both secondary and tertiary structures of the N-terminal domain resemble those of class I cytochrome c, displaying three α-helices and a compact overall folding. The C-terminal domain is less helical than the corresponding domain of Rs-DHC. The two heme groups are bridged by Tyr26 in correspondence with the shortest edge-to-edge distance, a feature which would facilitate fast internal electron transfer. The electronic properties of the two prosthetic centers are equivalent and sensitive to two acid–base equilibria with pK _a values of approximately 2.4 and 5, likely corresponding to protonation and detachment of the axial His ligands from the heme iron and a pH-linked conformational change of the protein, respectively. Reduction potentials of −0.144 and −0.257 V (vs. the standard hydrogen electrode), were determined for the C- and N-terminal heme groups, respectively. An approach based on the extended Debye–Hückel equation was applied for the first time to a two-centered metalloprotein and was found to reproduce successfully the ionic strength dependence of E°′.