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Mitochondria play an important role in numerous diseases as well as normative aging. Severe reduction in mitochondrial function contributes to childhood disorders such as Leigh Syndrome, whereas mild disruption can extend the lifespan of model organisms. The Caenorhabditis elegans isp-1 gene encodes the Rieske iron-sulfur protein subunit of cytochrome c oxidoreductase (complex III of the electron transport chain). The partial loss of function allele, isp-1(qm150), leads to several pleiotropic phenotypes. To better understand the molecular mechanisms of ISP-1 function, we sought to identify genetic suppressors of the delayed development of isp-1(qm150) animals. Here we report a series of intragenic suppressors, all located within a highly conserved six amino acid tether region of ISP-1. These intragenic mutations suppress all of the evaluated isp-1(qm150) phenotypes, including developmental rate, pharyngeal pumping rate, brood size, body movement, activation of the mitochondrial unfolded protein response reporter, CO2 production, mitochondrial oxidative phosphorylation, and lifespan extension. Furthermore, analogous mutations show a similar effect when engineered into the budding yeast Rieske iron-sulfur protein Rip1, revealing remarkable conservation of the structure-function relationship of these residues across highly divergent species. The focus on a single subunit as causal both in generation and in suppression of diverse pleiotropic phenotypes points to a common underlying molecular mechanism, for which we propose a "spring-loaded" model. These observations provide insights into how gating and control processes influence the function of ISP-1 in mediating pleiotropic phenotypes including developmental rate, movement, sensitivity to stress, and longevity. [ABSTRACT FROM AUTHOR]

We have used X-band ESEEM to study the reduced [2Fe-2S] cluster in adrenodoxin and Arthrospira platensisferredoxin. By use of a 2D approach (HYSCORE), we have shown that the cluster is involved in weak magnetic interactions with several nitrogens in each protein. Despite substantial differences in the shape and orientational dependence of individual cross-peaks, the major spectral features in both proteins are attributable to two peptide nitrogens (N1 and N2) with similar hyperfine couplings ∼1.1 and ∼0.70 MHz. The couplings determined represent a small fraction (0.0003–0.0005) of the unpaired spin density of the reduced cluster transferred to these nitrogens over H-bond bridges or the covalent bonds of cysteine ligands. Simulation of the HYSCORE spectra has allowed us to estimate the orientation of the nuclear quadrupole tensors of N1 and N2 in the g-tensor coordinate system. The most likely candidates for the role of N1 and N2 have been identified in the protein environment by comparing magnetic-resonance data with crystallographic structures of the oxidized proteins. A possible influence of redox-linked structural changes on ESEEM data is analyzed using available structures for related proteins in two redox states. [ABSTRACT FROM AUTHOR]

The rate of quinol oxidation by cytochrome bc1/b6f complex is in part associated with the redox potential ( Em) of its Rieske [2Fe-2S] center, for which an approximate correlation with the number of hydrogen bonds to the cluster has been proposed. Here we report comparative resonance Raman (RR) characterization of bacterial and archaeal high-potential Rieske proteins and their site-directed variants with a modified hydrogen bond network around the cluster. Major differences among their RR spectra appear to be associated in part with the presence or absence of Tyr-156 (in the Rhodobacter sphaeroides numbering) near one of the Cys ligands to the cluster. Elimination of the hydrogen bond between the terminal cysteinyl sulfur ligand (St) and Tyr-Oη (as with the Y156W variant, which has a modified histidine Nε p Ka,ox) induces a small structural bias of the geometry of the cluster and the surrounding protein in the normal coordinate system, and significantly affects some Fe-Sb/t stretching vibrations. This is not observed in the case of the hydrogen bond between the bridging sulfide ligand (Sb) and Ser-Oγ, which is weak and/or unfavorably oriented for extensive coupling with the Fe-Sb/t stretching vibrations. [ABSTRACT FROM AUTHOR]

The effect of different feeding behaviours of 1st and 4th instar Trichoplusia ni on photosynthesis of Arabidopsis thaliana var. Columbia was characterized using spatially resolved measurements of fluorescence and leaf temperature, as well as leaf gas exchange,. First instars made small holes with a large perimeter-to-area ratio and avoided veins, while 4th instars made large holes with a low perimeter-to-area ratio and consumed veins. Herbivory by 1st instars reduced photosynthesis more strongly in the remaining leaf tissue than that by 4th instars. Photosystem II operating efficiency (ΦPSII) was correlated with the rate of CO2 exchange, and reductions in ΦPSII in areas around the missing tissues contributed to a 15.6% reduction in CO2 assimilation on the first day following removal of 1st instars. The corresponding increases in non-photochemical quenching and greater rates of non-stomatal water loss from these regions, as well as the partial reversal of low ΦPSII by increasing the ambient CO2 concentration, suggests that localized water stress and reduced stomatal conductance contributed to the inhibition of photosynthesis. Damage by 1st but not 4th instars reduced the maximum quantum efficiency of photosystem II photochemistry (Fv/Fm) by 4–8%. While herbivory by both 1st and 4th instars increased dark respiration rates, the rates were too low to have contributed to the observed reductions in CO2 exchange. The small holes produced by 1st instars may have isolated patches of tissue from the vascular system thereby contributing to localized water stress. Since neither 1st nor 4th instar herbivory had a detectable effect on the expression of the Rubisco small subunit gene, the observed differences cannot be attributed to changes in expression of this gene. The mode of feeding by different instars of T. ni determined the photosynthetic response to herbivory, which appeared to be mediated by the level of water stress associated with herbivore damage. [ABSTRACT FROM PUBLISHER]

The bc1 complexes are intrinsic membrane proteins that catalyze the oxidation of ubihydroquinone and the reduction of cytochrome c in mitochondrial respiratory chains and bacterial photosynthetic and respiratory chains. The bc1 complex operates through a Q-cycle mechanism that couples electron transfer to generation of the proton gradient that drives ATP synthesis. Genetic defects leading to mutations in proteins of the respiratory chain, including the subunits of the bc1 complex, result in mitochondrial myopathies, many of which are a direct result of dysfunction at catalytic sites. Some myopathies, especially those in the cytochrome b subunit, exacerbate free-radical damage by enhancing superoxide production at the ubihydroquinone oxidation site. This bypass reaction appears to be an unavoidable feature of the reaction mechanism. Cellular aging is largely attributable to damage to DNA and proteins from the reactive oxygen species arising from superoxide and is a major contributing factor in many diseases of old age. An understanding of the mechanism of the bc1 complex is therefore central to our understanding of the aging process. In addition, a wide range of inhibitors that mimic the quinone substrates are finding important applications in clinical therapy and agronomy. Recent structural studies have shown how many of these inhibitors bind, and have provided important clues to the mechanism of action and the basis of resistance through mutation. This paper reviews recent advances in our understanding of the mechanism of the bc1 complex and their relation to these physiologically important issues in the context of the structural information available. [ABSTRACT FROM AUTHOR]

Discusses the function and diversity of cytochrome bc complexes. Purification of chloroplast and cyanobacterial bc complexes; Two-dimensional crystals and electron microscopy studies of cytochrome bc complexes; Parameters of different forms of mitochondrial cytochrome bc crystals.

Provides information on a study which identified pathways by which protons are released from the quinol-oxidizing site on quinol oxidation. Inhibitor and substrate complexes at the quinol-oxidizing site; Kinetic assays of substrate binding; Effects of mutation.

The ubiquinol: cytochrome-c oxidoreductase (cytochrome bc1 complex) is a central component of the mitochondrial respiratory chain as well as the respiratory and/or photosynthetic systems of numerous prokaryotic organisms. In Rhodobacter sphaeroides, the bc1 complex has a dual function. When the cells are grown photosynthetically, the bc1 complex is present in the intracytoplasmic membrane and is a critical component of the cyclic electron transport system. When the cells are grown in the dark in the presence of oxygen, the same bc1 complex is a necessary component of the cytochrome-c2-dependent respiratory chain. The fact that the bc1 complex from R. sphaeroides has been extensively studied, plus the ability to manipulate this organism genetically, makes this an ideal system for using site-directed mutagenesis to address questions relating to the structure and function of the bc1 complex. In the current work, the cloning and complete sequence of the fbc operon from R. sphaeroides is reported. As in other bacteria, this operon contains three genes, encoding the Rieske 2Fe-2S subunit, the cytochrome b subunit, and the cytochrome c1 subunit. Recombination techniques were used to delete the entire fbc operon from the chromosome. The resulting strain cannot grow photosynthetically, but can grow aerobically utilizing a quinol oxidase. Photosynthetic growth is restored by providing fbc operon on a plasmid, and the reappearance of the protein subunits and the spectroscopic features due to the bc1 complex are also demonstrated. Finally, a mutation is introduced within the gene encoding the cytochrome b subunit which is predicted to confer resistance to the inhibitor myxothiazol. It is shown that the resulting strain contains a functional bc1 complex which, as expected, is resistant to the inhibitor. Hence, this system is suitable for the detailed characterization of the bc1 complex, combining site-directed mutagenesis with the biochemical and biophysical techniques which have been previously developed for the study of photosynthetic bacteria. [ABSTRACT FROM AUTHOR]

Presents research which studied the X-ray crystal structure of the respiratory enzyme complex cytochrome bc1. Role of cytochrome bc1 in electron transfer; Study of the complex from chicken, cow and rabbit; Two different locations for the extrinsic domain of an enzyme component, an iron-sulphur protein; Nature of oxidation reaction mechanism.

1. The relation between the "high energy state" of photophosphorylation and delayed fluorescence has been studied in spinach chloroplasts. 2. In the presence of an electron acceptor or cofactors, the kinetics of the rise in intensity of light emitted i millisecond after illumination showed two distinct phases,—a rapid phase complete in less than 0.1 s, and a sigmoidal slow phase with a halfrise time of about 0.3 s. 3. The maximal intensity and time course of emission were found to vary with electron flow and the degree of coupling. With nigericin or with NH4Cl, the slow phase of the intensity rise was abolished but the rapid phase was unaffected. With valinomycin the rapid phase was substantially inhibited, but the extent of the slow phase was increased to give the same maximal intensity as observed in the absence of ionophore. With carbony cyanide p-trifluoromethoxyphenylhydrazone (FCCP), or a combination of valinomycm and nigericin, both phases were substantially inhibited. This spectrum of sensitivities has been interpreted as showing that the slow phase depends on the pH component, and a part of the rapid phase on the electrical component, of the electro-chemical H+-gradient. Addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea always inhibited emission. 4. A mechanism relating delayed fluorescence to the "high energy state" is suggested. The photochemical act of System II is envisaged as occurring between acceptor and donor sites on opposite sides of the thylakoid membrane and in equilibrium with pools the mid point potentials of which are dependent on the pH of the phase. It is shown that in such a system the energy conserved in the oxido reductive poise and in the electrochemical H+ gradient are additive and that both contribute to a decreased activation energy for delayed fluorescence. By dissipating the H+ gradient, the activation energy requirement is increased so that the intensity of emission falls. 5. It is concluded that other mechanisms for emission also operate, in particular one dependent on electron flow as such. [ABSTRACT FROM AUTHOR]

1. The shift of the carotenoid absorption spectrum in chromatophores prepared from Rhodopseudomonas spheroides induced by different types of actinic illumination has been resolved into several distinct, kinetic phases. A saturating 20 ns pulse from a Q-switched ruby laser elicited a spectral change more rapid than the response time of the measuring apparatus (1 µs). A rapid change 10-20% greater than this was produced during a saturating 200 µs xenon flash. Additional slower phases (half-risetimes approximately 300 µs and 5 ms) could be distinguished after the xenon flash. Continuous illumination produced a rapid change of similar extent, to that produced by xenon flash but with a risetime limited by the light intensity, and an equally extensive slow change of half-risetime approximately 83 ms. During prolonged illumination the carotenoid shift decayed by about 50% with an approximate half-decay time of 30 s. 2. The effects of certain electron-transport inhibitors, uncoupling agents, ion-transporting antibiotics and permeant anions on these phases and upon the decay after illumination have been investigated. None of these agents markedly affected the rapid phase of the shift induced by flash illumination. The relation of this phase to membrane potential was indicated by the fact that its decay was stimulated during electrophoretic ion flux. These observations are consistent with the suggestion that the rapid phase of the carotenoid shift is in response to separation of charge across the chromatophore membrane, between reaction centre bacteriochlorophyll and the primary acceptor. The effects of these specific chemical modifiers on the slower phases of the carotenoid shift were complex. They are discussed in terms of a model in which these phases reflect the generation of a membrane potential as a result of electrogenic oxidation-reduction processes. Antimycin A partly in.hired electron flow through such an electrogenic site(s). Valinomyein delayed the formation of the membrane potential by catalysing an initial, electrophoretic K+ efflux. Uncoupling agents caused a partial but, permanent collapse of the electrical field. Permeant anions produced a slight, lowering of the steady-state electrical field by raising the membrane conductivity. 3. The contribution of the membrane potential to the high-energy state leading to ATP synthesis is discussed. [ABSTRACT FROM AUTHOR]

1. A light-induced, energy dependent quenching of chlorophyll a fluorescence in the presence of 2,3,5,6-tetramethyl-p-phenylene diamine (diamino-durene) and 3-(3,4-dichlorophenyl)-l,1dimethylurea is described for isolated chloroplasts, and is related to the formation of the high-energy state of photophosphorylation. 2. The quenching is distinguished from the normal type, operative through the redox state of the Photosystem II quencher, by its light-intensity dependence, reversibility in the dark and sensitivity to uncoupling agents. 3. The sensitivity of the quenching to uncouplers and ionophorous antibiotics closely parallels that of photophosphorylation. 4. A similar quenching is described for other electron transport systems in the absence of dichlorophenyl-dimethylurea, when it is distinguished from Q-quenching on a time-basis. 5. The pH-optimum for the unncoupler-reversible quenching is about pH 8.0 for all electron flow systems used. This apparent discrepancy with the pH optimum for XE-formation is discussed. 6. Two other types of slow quenching seen during the Hill reaction with ferricyanide are described. One is found to be uncoupler enhanced and is related to acceleration of electron transport giving rise to a lower level of reduced Q Lq the steady state; the other is an irreversible loss of total fluorescence yield, which is most pronounced in coupled chloroplasts, and it is suggested that it is due to photochemical destruction of bulk pigment molecules via a side reaction of the excited state of chlorophyll. [ABSTRACT FROM AUTHOR]

Several intragenic mutations suppress theC. elegans isp-1(qm150)allele of the mitochondrial Rieske iron-sulfur protein (ISP), a catalytic subunit of Complex III of the respiratory chain. These mutations were located in a helical region of the “tether” span of ISP-1, distant from the primary mutation in the extrinsic head, and suppressed all pleiotropic phenotypes associated with theqm150allele. Analysis of these suppressors revealed control of electron transfer into Complex III through a “spring-loaded” mechanism involving a binding force for formation of enzyme-substrate complex, counter balanced by forces (a chemical “spring”) favoring helix formation in the tether. The primary P→S mutation results in inhibition of electron flow into the Q-cycle by decreasing the binding force, and the tether mutations relieve this inhibition by weakening the “spring.” In this commentary we discuss additional control features, and relate the primary inhibition to outcomes at the organismal level. In particular, the sensitivity to hyperoxia and the elevated reactive oxygen species (ROS) seen inisp-1(qm150), likely reflect over-reduction of the quinone pool, which is upstream of the inhibited site; at high O2, this would lead to increased ROS production through complex I. We speculate that alternative NADH:ubiquinone oxidoreductase activity inC. elegansfrom the worm apoptosis inducing factor (AIF) homolog (WAH-1) might also be involved, and that WAH-1 might have a “canary” function in detection of this adverse state (high O2/reduced pool), and a role in protection of the organism by transformation to AIF-like products, and apoptotic recycling of defective cells. [ABSTRACT FROM AUTHOR]