Complex structure of cytochrome c-cytochrome c oxidase reveals a novel protein-protein interaction mode.

Mitochondrial cytochrome c oxidase (CcO) transfers electrons from cytochrome c (Cyt.c) to O2 to generate H2O, a process coupled to proton pumping. To elucidate the mechanism of electron transfer, we determined the structure of the mammalian Cyt.c-CcO complex at 2.0-Å resolution and identified an electron transfer pathway from Cyt.c to CcO. The specific interaction between Cyt.c and CcO is stabilized by a few electrostatic interactions between side chains within a small contact surface area. Between the two proteins are three water layers with a long inter-molecular span, one of which lies between the other two layers without significant direct interaction with either protein. Cyt.c undergoes large structural fluctuations, using the interacting regions with CcO as a fulcrum. These features of the protein-protein interaction at the docking interface represent the first known example of a new class of protein-protein interaction, which we term "soft and specific". This interaction is likely to contribute to the rapid association/dissociation of the Cyt.c-CcO complex, which facilitates the sequential supply of four electrons for the O2 reduction reaction.

A unique respiratory adaptation in Drosophila independent of supercomplex formation.

Large assemblies of respiratory chain complexes, known as supercomplexes, are present in the mitochondrial membrane in mammals and yeast, as well as in some bacterial membranes. The formation of supercomplexes is thought to contribute to efficient electron transfer, stabilization of each enzyme complex, and inhibition of reactive oxygen species (ROS) generation. In this study, mitochondria from various organisms were solubilized with digitonin, and then the solubilized complexes were separated by blue native PAGE (BN-PAGE). The results revealed a supercomplex consisting of complexes I, III, and IV in mitochondria from bovine and porcine heart, and a supercomplex consisting primarily of complexes I and III in mitochondria from mouse heart and liver. However, supercomplexes were barely detectable in Drosophila flight-muscle mitochondria, and only dimeric complex V was present. Drosophila mitochondria exhibited the highest rates of oxygen consumption and NADH oxidation, and the concentrations of the electron carriers, cytochrome c and quinone were higher than in other species. Respiratory chain complexes were tightly packed in the mitochondrial membrane containing abundant phosphatidylethanolamine with the fatty acid palmitoleic acid (C16:1), which is relatively high oxidation-resistant as compared to poly-unsaturated fatty acid. These properties presumably allow efficient electron transfer in Drosophila. These findings reveal the existence of a new mechanism of biological adaptation independent of supercomplex formation.

Solubilization conditions for bovine heart mitochondrial membranes allow selective purification of large quantities of respiratory complexes I, III, and V.

Ascertaining the structure and functions of mitochondrial respiratory chain complexes is essential to understanding the biological mechanisms of energy conversion; therefore, numerous studies have examined these complexes. A fundamental part of that research involves devising a method for purifying samples with good reproducibility; the samples obtained need to be stable and their constituents need to retain the same structure and functions they possess when in mitochondrial membranes. Submitochondrial bovine heart particles were isolated using differential centrifugation to adjust to a membrane concentration of 46.0% (w/v) or 31.5% (w/v) based on weight. After 0.7% (w/v) deoxycholic acid, 0.4% (w/v) decyl maltoside, and 7.2% (w/v) potassium chloride were added to the mitochondrial membranes, those membranes were solubilized. At a membrane concentration of 46%, complex V was selectively solubilized, whereas at a concentration of 31.5% (w/v), complexes I and III were solubilized. Two steps-sucrose density gradient centrifugation and anion-exchange chromatography on a POROS HQ 20 µm column-enabled selective purification of samples that retained their structure and functions. These two steps enabled complexes I, III, and V to be purified in two days with a high yield. Complexes I, III, and V were stabilized with n-decyl-ß-D-maltoside. A total of 200 mg-300 mg of those complexes from one bovine heart (1.1 kg muscle) was purified with good reproducibility, and the complexes retained the same functions they possessed while in mitochondrial membranes.

Purification of Active Respiratory Supercomplex from Bovine Heart Mitochondria Enables Functional Studies.

To understand the roles of mitochondrial respiratory chain supercomplexes, methods for consistently separating and preparing supercomplexes must be established. To this end, we solubilized supercomplexes from bovine heart mitochondria with digitonin and then replaced digitonin with amphipol (A8-35), an amphiphilic polymer. Afterward, supercomplexes were separated from other complexes by sucrose density gradient centrifugation. Twenty-six grams of bovine myocardium yielded 3.2 mg of amphipol-stabilized supercomplex. The purified supercomplexes were analyzed based on their absorption spectra as well as Q10 (ubiquinone with ten isoprene units) and lipid assays. The supercomplex sample did not contain cytochrome c but did contain complexes I, III, and IV at a ratio of 1:2:1, 6 molecules of Q10, and 623 atoms of phosphorus. When cytochrome c was added, the supercomplex exhibited KCN-sensitive NADH oxidation; thus, the purified supercomplex was active. Reduced complex IV absorbs at 444 nm, so we measured the resonance Raman spectrum of the reduced amphipol-solubilized supercomplex and the mixture of amphipol-solubilized complexes I1, III2, and IV1 using an excitation wavelength of 441.6 nm, allowing measurement precision comparable with that obtained for complex IV alone. Use of the purified active sample provides insights into the effects of supercomplex formation.

A highly durable, stretchable, transparent and conductive carbon nanotube-polymeric acid hybrid film.

We demonstrate a highly durable and stretchable carbon nanotube (CNT)-polymer transparent conductive film by utilizing polyacrylic acid wrapping to simultaneously disperse and dope CNTs. This novel strategy not only enables an easy and industrially scalable process where neither removal of dispersant nor further doping is necessary, but also results in a boost in the conductivity and durability, and correspondingly it provides infinite versatility for various applications. A significant improvement in conductivity being comparable to acid doping is achieved through a hybrid structure in which a polyacrylic acid monolayer helically and partially wraps the CNT surface. The optimum films show sheet resistances of 150 and 60 Ω per square at 91% and 84% optical transmittances, respectively. Notably, the sheet resistances of hybrid films remain almost constant after aging tests under conditions of 85 °C and 85% relative humidity for more than 3000 h, and bending and stretching tests of more than 1 million cycles, suggesting outstanding long-term stability and record-high flexibility and stretchability. Therefore, the highly transparent conductive film, combined with excellent long-term and mechanical durability, and surprisingly easy processability, opens new routes for next-generation wearable electronics and bioelectronics.

Structure of bovine cytochrome c oxidase in the ligand-free reduced state at neutral pH.

Cytochrome c oxidase (CcO), the terminal oxidase in cellular respiration, couples proton pumping to O2 reduction. Mammalian CcO resides in the inner mitochondrial membrane. Previously, a model of H-pathway proton pumping was proposed based on various CcO crystal structures. However, all previously determined structures were solved using crystals obtained at pH 5.7, which differs from the environmental pH of CcO in the inner membrane. The structures of fully oxidized and ligand-free reduced CcO at pH 7.3 have now been determined. Structural comparison between the oxidized and reduced states revealed that the structural alterations that occurred upon redox change at pH 5.7 in Asp51, the magnesium-containing cluster, haem groups and helix X, which provide important structural evidence for the H-pathway proton-pumping proposal, also occur at pH 7.3. These structural alterations were restricted to a local region of CcO; no domain movement was detected, nor were significant structural alterations detected in peripheral regions at either pH value. These observations indicate that the small and precise structural alterations that occur over the course of the reaction cycle are not affected by pH change, and that isolated CcO precisely performs proton pumping via the H-pathway over a wide pH range. Because the pH is not uniform across the molecular surface of CcO, the fact that the overall structure of CcO is not affected by pH changes ensures the high enzymatic efficiency of this protein in the mitochondria.

Structure of bovine cytochrome c oxidase crystallized at a neutral pH using a fluorinated detergent.

Cytochrome c oxidase (CcO) couples proton pumping to O2 reduction. Its enzymatic activity depends sensitively on pH over a wide range. However, owing to difficulty in crystallizing this protein, X-ray structure analyses of bovine CcO aimed at understanding its reaction mechanism have been conducted using crystals prepared at pH 5.7, which is significantly lower than that in the cell. Here, oxidized CcO at pH 7.3 was crystallized using a fluorinated octyl-maltoside derivative, and the structure was determined at 1.77 Šresolution. No structural differences between crystals obtained at the neutral pH and the acidic pH were detected within the molecules. On the other hand, some differences in intermolecular interactions were detected between the two types of crystal. The influence of pH on the molecular surface is likely to contribute to the pH dependency of the aerobic oxidation of ferrocytochrome c.

GraDeR: Membrane Protein Complex Preparation for Single-Particle Cryo-EM.

We developed a method, named GraDeR, which substantially improves the preparation of membrane protein complexes for structure determination by single-particle cryo-electron microscopy (cryo-EM). In GraDeR, glycerol gradient centrifugation is used for the mild removal of free detergent monomers and micelles from lauryl maltose-neopentyl glycol detergent stabilized membrane complexes, resulting in monodisperse and stable complexes to which standard processes for water-soluble complexes can be applied. We demonstrate the applicability of the method on three different membrane complexes, including the mammalian FoF1 ATP synthase. For this highly dynamic and fragile rotary motor, we show that GraDeR allows visualizing the asymmetry of the F1 domain, which matches the ground state structure of the isolated domain. Therefore, the present cryo-EM structure of FoF1 ATP synthase provides direct structural evidence for Boyer's binding change mechanism in the context of the intact enzyme.

Water disinfection by electrochemical treatment.

The electrochemical disinfection of germinated brown rice (GBR) circulating water and cooling tower water containing Legionella bacteria was investigated. Results showed the total aerobic plate counts (APC) in the treated GBR circulating water decreased significantly and the turbidity was largely improved at a pulse voltage of 1.0 kV; Legionella bacteria were also disinfected effectively at 1.0 kV. The disinfection was attributed to the synergistic effects of the oxide anode, the electric field, and the radicals formed during the electrochemical treatment. This suggests that electrochemical treatment could be applicable to the disinfection of water from other sources.

Development of a high performance electrochemical wastewater treatment system.

In order to construct a high performance electrochemical system for practical use in industrial and municipal wastewater treatment, laboratory scale electrochemical experiments were performed to select anode materials by applying pulse voltage. Based on the results obtained from laboratory experiments, a pilot plant of electrochemical treatment system (0.3 m3 h(-1)) was successfully developed, in which electrocoagulation and electrooxidation processes were used. The performance of the treatment system was evaluated by treating domestic wastewater, pond water containing algae and wastewater from hog raising. As a result, production of hydroxyl radicals detected with p-nitrosodimethylaniline (RNO) at Ti/RuO(2)-TiO(2) anode was larger than with a platinum anode, and hydroxyl radicals were not detected at Ti anode. Moreover, a significant difference in electrocatalytic properties for ammonia oxidation between platinum and Ti/RuO(2)-TiO(2) electrodes was not observed from the cyclic voltammogram. The removal of T-N, T-P, NH(4)-N and COD from domestic wastewater and pond water containing algae was approximately 90%, while the removal of chlorophyll-a (chl-a) of algae was approximately 100%. Although the electrochemical treatment system was effective on biologically treated wastewater from hog raising, the treatment of raw wastewater was not remarkable. Therefore, the electrochemical treatment system requires pretreatment when used with wastewater containing high concentrations of suspended solids.

Three-dimensional structure of bovine heart NADH: ubiquinone oxidoreductase (complex I) by electron microscopy of a single negatively stained two-dimensional crystal.

Bovine heart NADH:ubiquinone oxidoreductase (complex I), which is the largest (about 1 MDa) membrane protein complex in the mitochondrial respiratory chain, catalyzes the electron transfer from NADH to ubiquinone, coupled with proton pumping. We have crystallized bovine complex I in reconstituted lipid bilayers and obtained a three-dimensional density map by the electron crystallographic analysis of a single negatively stained two-dimensional crystal. The asymmetric unit with dimensions of a = 388 Å, b = 129 Å and γ = 90° contains two molecules and is of P1 symmetry. Structural differences between the two molecules indicate flexibility of the hydrophilic domain relative to the membrane-embedded domain.

Solid-state light-emitting devices based on the tris-chelated ruthenium(II) complex. 4. High-efficiency light-emitting devices based on derivatives of the tris(2,2'-bipyridyl) ruthenium(II) complex.

Light-emitting devices from the tris(2,2'-bipyridyl)ruthenium(II) complex [Ru(bpy)(3)(2+)] and new derivatives thereof were prepared. Due to the electrochemical nature of the device operation, single-layer devices in an ITO/ Ru(bpy)(3)(2+) complex + PMMA/Ag sandwich configuration achieved very high external quantum efficiencies. The derivatives of the Ru(bpy)(3)(2+) complex were designed and synthesized to inhibit self-quenching of the excited state by adding different alkyl substituents on the bipyridyl ligands. As a result, devices that contain these new Ru(bpy)(3)(2+) complexes show a higher photoluminescence and electroluminescence efficiency than devices made from the unmodified Ru(bpy)(3)(2+) complex. External quantum efficiencies up to 5.5% at brightnesses in the range of 10-50 cd/m(2) are reported. In addition, the response time of such devices (which is a result of the electrochemical operation) has been shortened dramatically. An "instantaneous" light emission is achieved for devices that employ smaller counterions such as BF(4)(-) to increase the ionic conductivity. Such a device shows a response time of less than 1 s to emit 10-20 cd/m(2) after the operating voltage of 2.4 V has been applied.