Iba57p participates in maturation of a [2Fe-2S]-cluster Rieske protein and in formation of supercomplexes III/IV of Saccharomyces cerevisiae electron transport chain.

The [Fe-S] late-acting subsystem comprised of Isa1p/Isa2p, Grx5p, and Iba57p proteins (Fe-S-IBG subsystem) is involved in [4Fe-4S]-cluster protein assembly. The effect of deleting IBA57 in Saccharomyces cerevisiae on mitochondrial respiratory complex integration and functionality associated with Rieske protein maturation was evaluated. The iba57Δ mutant showed decreased expression and maturation of the Rieske protein. The loss of Rieske protein caused by IBA57 deletion affected the structure of supercomplexes III2IV2 and III2IV1 and their integration into the mitochondria, causing dysfunction in the electron transport chain. These effects were correlated with decreased cytochrome functionality and content in the iba57Δ mutant. These findings suggest that Iba57p participates in maturation of the [2Fe-2S]-cluster into the Rieske protein and that Rieske protein plays important roles in the conformation and functionality of mitochondrial supercomplex III/IV in the electron transport chain.

The cytochrome bd-I respiratory oxidase augments survival of multidrug-resistant Escherichia coli during infection.

Nitric oxide (NO) is a toxic free radical produced by neutrophils and macrophages in response to infection. Uropathogenic Escherichia coli (UPEC) induces a variety of defence mechanisms in response to NO, including direct NO detoxification (Hmp, NorVW, NrfA), iron-sulphur cluster repair (YtfE), and the expression of the NO-tolerant cytochrome bd-I respiratory oxidase (CydAB). The current study quantifies the relative contribution of these systems to UPEC growth and survival during infection. Loss of the flavohemoglobin Hmp and cytochrome bd-I elicit the greatest sensitivity to NO-mediated growth inhibition, whereas all but the periplasmic nitrite reductase NrfA provide protection against neutrophil killing and promote survival within activated macrophages. Intriguingly, the cytochrome bd-I respiratory oxidase was the only system that augmented UPEC survival in a mouse model after 2 days, suggesting that maintaining aerobic respiration under conditions of nitrosative stress is a key factor for host colonisation. These findings suggest that while UPEC have acquired a host of specialized mechanisms to evade nitrosative stresses, the cytochrome bd-I respiratory oxidase is the main contributor to NO tolerance and host colonisation under microaerobic conditions. This respiratory complex is therefore of major importance for the accumulation of high bacterial loads during infection of the urinary tract.

The Terminal Oxidase Cytochrome bd Promotes Sulfide-resistant Bacterial Respiration and Growth.

Hydrogen sulfide (H2S) impairs mitochondrial respiration by potently inhibiting the heme-copper cytochrome c oxidase. Since many prokaryotes, including Escherichia (E.) coli, generate H2S and encounter high H2S levels particularly in the human gut, herein we tested whether bacteria can sustain sulfide-resistant O2-dependent respiration. E. coli has three respiratory oxidases, the cyanide-sensitive heme-copper bo3 enzyme and two bd oxidases much less sensitive to cyanide. Working on the isolated enzymes, we found that, whereas the bo3 oxidase is inhibited by sulfide with half-maximal inhibitory concentration IC50 = 1.1 ± 0.1 µM, under identical experimental conditions both bd oxidases are insensitive to sulfide up to 58 µM. In E. coli respiratory mutants, both O2-consumption and aerobic growth proved to be severely impaired by sulfide when respiration was sustained by the bo3 oxidase alone, but unaffected by ≤200 µM sulfide when either bd enzyme acted as the only terminal oxidase. Accordingly, wild-type E. coli showed sulfide-insensitive respiration and growth under conditions favouring the expression of bd oxidases. In all tested conditions, cyanide mimicked the functional effect of sulfide on bacterial respiration. We conclude that bd oxidases promote sulfide-resistant O2-consumption and growth in E. coli and possibly other bacteria. The impact of this discovery is discussed.

A combined electrochemical and optical trapping platform for measuring single cell respiration rates at electrode interfaces.

Metal-reducing bacteria gain energy by extracellular electron transfer to external solids, such as naturally abundant minerals, which substitute for oxygen or the other common soluble electron acceptors of respiration. This process is one of the earliest forms of respiration on earth and has significant environmental and technological implications. By performing electron transfer to electrodes instead of minerals, these microbes can be used as biocatalysts for conversion of diverse chemical fuels to electricity. Understanding such a complex biotic-abiotic interaction necessitates the development of tools capable of probing extracellular electron transfer down to the level of single cells. Here, we describe an experimental platform for single cell respiration measurements. The design integrates an infrared optical trap, perfusion chamber, and lithographically fabricated electrochemical chips containing potentiostatically controlled transparent indium tin oxide microelectrodes. Individual bacteria are manipulated using the optical trap and placed on the microelectrodes, which are biased at a suitable oxidizing potential in the absence of any chemical electron acceptor. The potentiostat is used to detect the respiration current correlated with cell-electrode contact. We demonstrate the system with single cell measurements of the dissimilatory-metal reducing bacterium Shewanella oneidensis MR-1, which resulted in respiration currents ranging from 15 fA to 100 fA per cell under our measurement conditions. Mutants lacking the outer-membrane cytochromes necessary for extracellular respiration did not result in any measurable current output upon contact. In addition to the application for extracellular electron transfer studies, the ability to electronically measure cell-specific respiration rates may provide answers for a variety of fundamental microbial physiology questions.

Mitochondrial dysfunction resulting from the absence of mitochondrial porin in Neurospora crassa.

Porin, the voltage-dependent anion-selective channel (VDAC) in the mitochondrial outer membrane, contributes to metabolism and apoptosis. VDAC function was investigated in Neurospora, an obligate aerobe with a single porin. Porinless strains are viable, with cold-sensitive growth, cytochrome deficiencies and overexpression of alternative oxidase. iTRAQ labeling of mitochondria from a porinless strain and its progenitor revealed a small group of proteins with altered expression levels in the mutant organelles. Porinless Neurospora appears to compensate not by inducing alternative pores, but by altering electron flow and nucleotide metabolism. Transcriptional and post-transcriptional mechanisms contribute to the response, reflecting the extent of porin influence.

Involvement and specificity of Shewanella oneidensis outer membrane cytochromes in the reduction of soluble and solid-phase terminal electron acceptors.

The formation of outer membrane (OM) cytochromes seems to be a key step in the evolution of dissimilatory iron-reducing bacteria. They are believed to be the endpoints of an extended respiratory chain to the surface of the cell that establishes the connection to insoluble electron acceptors such as iron or manganese oxides. The gammaproteobacterium Shewanella oneidensis MR-1 contains the genetic information for five putative OM cytochromes. In this study, the role and specificity of these proteins were investigated. All experiments were conducted using a markerless deletion mutant in all five OM cytochromes that was complemented via the expression of single, plasmid-encoded genes. MtrC and MtrF were shown to be potent reductases of chelated ferric iron, birnessite, and a carbon anode in a microbial fuel cell. OmcA-producing cells were unable to catalyze iron and electrode reduction, although the protein was correctly produced and oriented. However, OmcA production resulted in a higher birnessite reduction rate compared with the mutant. The presence of the decaheme cytochrome SO_2931 as well as the diheme cytochrome SO_1659 did not rescue the phenotype of the deletion mutant.

Further evidence for the role of cryptochromes in retinohypothalamic photoreception/phototransduction.

Cryptochrome is a blue-light absorbing photopigment that has been proposed to act as a photoreceptor for a variety of nonvisual light-responsive tasks. While mouse models have suggested an important role for cryptochrome in nonvisual photoreception, there are no biochemical data demonstrating the functional photoreceptive capability of cryptochrome in mice. There are two models that describe the effect of cryptochrome on light responsive events: (1) cryptochrome is a photoreceptor or (2) cryptochrome is required for either normal phototransduction from the retina to the brain or for normal transcriptional regulation in the brain, irrespective of light. To differentiate between these two models, we have examined the integrity of the regulatory mechanism of c-fos in cryptochromeless cell lines and in the suprachiasmatic nucleus (SCN) of cryptochromeless mice. Photoinduction of c-fos mRNA in the SCN can be used as a marker for circadian photoreception/phototransduction and it is drastically reduced in mice lacking cryptochromes. Our results indicate that light-independent transcription regulatory system of c-fos is normal in cryptochromeless mice and that the reduced c-fos light responsiveness in the absence of cryptochromes is due to a loss of photoreceptor function.

Role of mitochondrial dysfunction in Alzheimer's disease.

Abnormalities in mitochondrial function relate to the spectrum of pathological changes seen in Alzheimer's disease. Here we review the causes and consequences of mitochondrial disturbances in Alzheimer's disease as well as how this information might impact on therapeutic approaches to this disease.

Genetic analysis of chloroplast c-type cytochrome assembly in Chlamydomonas reinhardtii: One chloroplast locus and at least four nuclear loci are required for heme attachment.

Chloroplasts contain up to two c-type cytochromes, membrane-anchored cytochrome f and soluble cytochrome c6. To elucidate the post-translational events required for their assembly, acetate-requiring mutants of Chlamydomonas reinhardtii that have combined deficiencies in both plastid-encoded cytochrome f and nucleus-encoded cytochrome c6 have been identified and analyzed. For strains ct34 and ct59, where the phenotype displays uniparental inheritance, the mutations were localized to the chloroplast ccsA gene, which was shown previously to be required for heme attachment to chloroplast apocytochromes. The mutations in another eight strains were localized to the nuclear genome. Complementation tests of these strains plus three previously identified strains of the same phenotype (ac206, F18, and F2D8) indicate that the 11 ccs strains define four nuclear loci, CCS1-CCS4. We conclude that the products of the CCS1-CCS4 loci are not required for translocation or processing of the preproteins but, like CcsA, they are required for the heme attachment step during assembly of both holocytochrome f and holocytochrome c6. The ccsA gene is transcribed in each of the nuclear mutants, but its protein product is absent in ccs1 mutants, and it appears to be degradation susceptible in ccs3 and ccs4 strains. We suggest that Ccsl may be associated with CcsA in a multisubunit "holocytochrome c assembly complex," and we hypothesize that the products of the other CCS loci may correspond to other subunits.

Isolation of a cytochrome-deficient mutant strain of Sporomusa sphaeroides not capable of oxidizing methyl groups.

The homoacetogenic anaerobic bacterium Sporomusa sphaeroides was mutagenized with UV light. Taking advantage of the ampicillin enrichment technique and a newly developed test for the detection of heme in bacterial colonies, the cytochrome-deficient mutant strain S. sphaeroides BK824 was isolated. In contrast to the wild type, this mutant strain failed to grow on betaine, betaine plus methanol, H2 plus CO2, and methanol plus CO2. Growth on betaine plus formate, betaine plus H2, betaine plus pyruvate, methanol plus H2 and CO2, and acetoin was not impaired. All enzymes of the Wood pathway as well as hydrogenase and carbon monoxide dehydrogenase were detectable at comparable activities in both the wild type and the cytochrome-deficient mutant. Labeling experiments with [14C]methanol demonstrated the inability of S. sphaeroides BK824 to oxidize methyl groups. The role of cytochromes in electron transport steps associated with the Wood pathway enzymes and their possible role in energy conservation during autotrophic growth in acetogens are discussed.

[Mitochondrial myopathies and encephalomyopathies. Neuromuscular and central nervous system diseases caused by defects in mitochondrial oxidative metabolism]. / Mitochondriale Myopathien und Enzephalomyopathien. Neuromuskuläre und zentralnervöse Erkrankungen infolge von Defekten des mitochondrialen oxydativen Stoffwechsels.

In this review the clinical data and biochemical findings of disorders of oxidative metabolism in the mitochondria are summarized. Defects in pyruvate metabolism, the citric acid circle, the respiratory chain and less well defined disorders of energy transfer are discussed.

Organic acids in urine of patients with congenital lactic acidoses: an aid to differential diagnosis.

The differential diagnosis of patients with apparent congenital lactic acidoses poses one of the most intractable problems in the study of patients with disorders of organic acid metabolism. An outline of the factors leading to a lactic acidosis, particularly in infants and young children, together with a brief review of the known causes of congenital lactic acidosis, are presented. Quantitative examination of the organic acids excreted by patients with proven enzyme deficiencies causing congenital lactic acidosis has demonstrated the characteristic patterns that are associated with specific disorders of this kind. After exclusion of uninherited, acquired and secondary metabolic causes of lactic acidosis, the quantitative patterns of organic acid excretion, together with other clinical and biochemical observations, provide valuable indicators of the area of the underlying primary metabolic disorder for subsequent selected, confirmatory, enzymology. The study of organic acids has a key and central role in the approach to the clinical and biochemical investigation and diagnosis of patients with congenital lactic acidoses.

Deficiency of cytochromes b and aa3 in muscle from a floppy infant with cytochrome oxidase deficiency.

A girl was presented suffering from generalised weakness and cardiorespiratory insufficiency. She succumbed at the age of 5 months. Lactate levels were elevated in serum, cerebrospinal fluid and urine. Histopathological examination revealed a mitochondrial myopathy. In muscle tissue the cytochrome oxydase activity was strongly reduced. The content of cytochromes b and aa3 was very low. At autopsy a cardiomyopathy was found.

Progressive poliodystrophy (Alpers' disease) with a defect in cytochrome aa3 in muscle: a report of two unrelated patients.

We present two unrelated patients, a boy and a girl, with a progressive neurologic disorder, characterized by psychomotor retardation, seizures and paresis, the illness being exacerbated during stressful periods. Lactate levels in serum and cerebrospinal fluid were elevated in both patients. Histopathologic studies of muscle tissue revealed mitochondrial abnormalities in the boy; in the girl, slight neuronal degeneration was observed. A cerebral biopsy in the girl showed abnormalities compatible with progressive poliodystrophy. Autopsy in the boy demonstrated progressive poliodystrophy. Biochemical studies in muscle tissue showed a defect of cytochrome aa3 in both patients, connected with a defect of cytochrome b in the girl. The association of defective pyruvate metabolism and progressive poliodystrophy is discussed.

Light effects in yeast: relation between the respiratory deficiency and light sensitivity in yeast.

Visible light of 5,000 lux intensity has been shown to photokill yeast cells at 12 degrees C. In the present report some of isogenic respiratory deficient mit- and nuclear mutants were compared for their sensitivity to light. No close correlation between the cytochromes spectra and light resistance was observed. Although, the nuclear and rho- mutants which lack cytochromes a + a3 and b are as a rule light resistant. Photokilling effect in yeast seems to be dependent both on the sufficiency of respiratory chain and on protein synthesis probably on cytoplasmic level.

Aminoglycoside-resistant mutants of Pseudomonas aeruginosa deficient in cytochrome d, nitrite reductase, and aerobic transport.

Two gentamicin-resistant mutants of Pseudomonas aeruginosa PAO 503 were selected after ethyl methane sulfonate mutagenesis. Mutant PAO 2403 had significantly increased resistance to aminoglycoside but not to other antibiotics. Mutant PAO 2402 showed a similar spectrum of resistance but of lower magnitude. Both mutants showed no detectable cytochrome d and had a high frequency of reversion to a fully wild-type phenotype. PAO 2403 had a marked decrease and PAO 2402 had a moderate decrease in nitrite reductase activity. Both mutants had reduced uptake of gentamicin and dihydrostreptomycin. Mutant PAO 2403 showed a general decrease in transport rate of cationic compounds, whereas mutant PAO 2402 had only deficient glucose transport. Both mutants showed enhanced rates of glutamine transport and no change in glutamic acid transport. Other components of electron transport and oxidative phosphorylation were normal. These mutants involve ferrocytochrome C551 oxidoreductase formed only on anaerobic growth but illustrate transport defects in aerobically grown cells.

[Selection method and the characteristics of a cytochrome (a+a3)-deficient mutant of Candida parapsilosis yeasts]. / Sposob otbora i kharakteristika tsitokhrom (a+a3)-defitsitnogo mutanta drozhzhei Candida parapsilosis.

A method was developed for selecting cytochrome-deficient mutants of yeast Candida parapsilosis; the method is based on determining the rate of inhibition of oxygen uptake by benzhydroxamic acid, an inhibitor of cyanide-resistant oxidase in the cells of preliminarily obtained yeast mutants. The mutant (C. parapsilosis bhas-1) lacks cytochrome a+a3, contains the same quantity of cytochrome b as the wild strain and a twice as low quantity of cytochrome c. In contrast to the wild strain, the mutant does not grow on ethanol for the first two days but, being cultivated further (up to 5 days), in a medium with ethanol, it resumes its capability to utilize ethanol as a carbon and energy source. Prolonged cultivation in the medium with ethanol induces the biosynthesis of cytochrome oxidase while cytochrome a+a3 is not induced in a medium supplied with glucose. The biomass accumulated by the mutant in the medium with glucose is twice as low comparing to that of the wild strain. The oxidative activity of the mutant mitochondria involves cyanide, resistant oxidase. The mitochondria of the mutant oxidize NAD-dependent substrates, NADH, NADPH, succinate, alpha-glycerophosphate, ethanol and lactate. The mitochondria have a low respiratory control and phosphorylate ADP while oxidizing NAD-dependent substrates, ethanol and lactate, but not alpha-glycerophosphate, succinate, NADH and NADPH.

The orientation of iron-sulphur clusters in membrane multilayers prepared from aerobically-grown Escherichia coli K12 and a cytochrome-deficient mutant.

1. Membrane particles prepared from ultrasonically-disrupted, aerobically-grown Escherichia coli were centrifuged on to a plastic film that was supported perpendicular to the centrifugal field to yield oriented membrane multilayers. In such preparations, there is a high degree of orientation of the planes of the membranes such that they lie parallel to each other and to the supporting film. 2. When dithionite- or succinate-reduced multilayers are rotated in the magnetic field of an e.p.r. spectrometer, about an axis lying in the membrane plane, angular-dependent signals from an iron-sulphur cluster at g(x)=1.92, g(y)=1.93 and g(z)=2.02 are seen. The g=1.93 signal has maximal amplitude when the plane of the multilayer is perpendicular to the magnetic field. Conversely, the g=2.02 signal is maximal when the plane of the multilayer is parallel with the magnetic field. 3. Computer simulations of the experimental data show that the cluster lies in the cytoplasmic membrane with the g(y) axis perpendicular to the membrane plane and with the g(x) and g(z) axes lying in the membrane plane. 4. In partially-oxidized multilayers, a signal resembling the mitochondrial high-potential iron-sulphur protein (Hipip) is seen whose g(z)=2.02 axis may be deduced as lying perpendicular to the membrane plane. 5. Appropriate choice of sample temperature and receiver gain reveals two further signals in partially-reduced multilayers: a g=2.09 signal arises from a cluster with its g(z) axis in the membrane plane, whereas a g=2.04 signal is from a cluster with the g(z) axis lying along the membrane normal. 6. Membrane particles from a glucose-grown, haem-deficient mutant contain dramatically-lowered levels of cytochromes and exhibit, in addition to the iron-sulphur clusters seen in the parental strain, a major signal at g=1.90. 7. Only the latter may be demonstrated to be oriented in multilayer preparations from the mutant. 8. Comparisons are drawn between the orientations of the iron-sulphur proteins in the cytoplasmic membrane of E. coli and those in mitochondrial membranes. The effects of diminished cytochrome content on the properties of the iron-sulphur proteins are discussed.