Active-Site Structure of the Thermophilic Foc-Subunit Ring in Membranes Elucidated by Solid-State NMR

F_oF₁-ATP synthase uses the electrochemical potential across membranes or ATP hydrolysis to rotate the F_oc-subunit ring. To elucidate the underlying mechanism, we carried out a structural analysis focused on the active site of the thermophilic c-subunit (TF_oc) ring in membranes with a solid-state NMR method developed for this purpose. We used stereo-array isotope labeling (SAIL) with a cell-free system to highlight the target. TF_oc oligomers were purified using a virtual ring His tag. The membrane-reconstituted TF_oc oligomer was confirmed to be a ring indistinguishable from that expressed in E. coli on the basis of the H⁺-translocation activity and high-speed atomic force microscopic images. For the analysis of the active site, 2D ¹³C-¹³C correlation spectra of TF_oc rings labeled with SAIL-Glu and -Asn were recorded. Complete signal assignment could be performed with the aid of the C^α_i+1-C^α_i correlation spectrum of specifically ¹³C,¹⁵N-labeled TF_oc rings. The C^δ chemical shift of Glu-56, which is essential for H⁺ translocation, and related crosspeaks revealed that its carboxyl group is protonated in the membrane, forming the H⁺-locked conformation with Asn-23. The chemical shift of Asp-61 C^γ of the E. coli c ring indicated an involvement of a water molecule in the H⁺ locking, in contrast to the involvement of Asn-23 in the TF_oc ring, suggesting two different means of proton storage in the c rings.     

Studies on the characterization of the Rho(D) antigen

The Rh_o(D) antigen of red cell membranes was solubilized using ethylenediamine tetraacetic acid (EDTA) and 2-mercaptoethanol. The solubilized antigen was partially separated from other solubilized membrane components using molecular filtration. The antigen was treated with various enzymes to learn some of the chemical characteristics. It was found that the activity of the antigen, as measured by hemagglutination inhibition, was not affected by bee venom phospholipase A, Clostridium welchii phospholipase C, calf-intestinal alkaline phosphatase, Vibrio cholerae neuraminidase, pig kidney leucine aminopeptidase, bovine pancreatic carboxypeptidase A, a pig pancreatic carboxypeptidase B. However, the proteolytic enzymes, pronase, trypsin, chymotrypsin and papain, did destroy Rh_o(D) activity as measured by hemagglutination inhibition. These results indicate that protein is an important part of the active determinant of the Rh_o(D) antigen. The experiments by other investigators have shown that lipid is important to maintain the Rh_o(D) activity in the intact membrane; lipid probably helps to maintain the structural conformation of the Rh_o(D) molecule in its natural environment. The solubilized Rh_o(D) molecules are apparently not dependent on lipid for their Rh_o(D) activity.     

CO-binding pigments and the functional terminal oxidase of the trypanosomatid hemoflagellate Crithidia fasciculata

CO-difference absorbance spectra of both intact cells and of mitochondrial preparations isolated from Crithidia fasciculata were obtained after anaerobiosis was attained either with substrates or with dithionite. Under both sets of conditions, the CO-difference spectrum of cytochrome a₃, with difference absorbance maxima at 430 and 589 nm and minima at 443 and 612 nm, was readily identified in both the intact cells and in the mitochondria. In addition to the difference absorbance bands of cytochrome a₃-CO, three difference absorbance maxima at 417, 538 and 570 nm and a minimum at 556 nm were observed. The magnitude of the maximum at 570 nm relative to the maximum of cytochrome a₃-CO at 589 nm was less for mitochondria rendered anaerobic with substrate than for mitochondria rendered anaerobic with dithionite. This difference was taken to define operationally two groups of mitochondrial CO-binding pigments: Group I is that group observed on anaerobiosis with substrate: Group II is the additional group observed on anaerobiosis with dithionite. The Group I CO-binding pigments were virtually absent from submitochondrial particles derived by sonication, but the Group II pigments remained.Photochemical action spectra were obtained with isolated mitochondria and intact cells to ascertain if cytochrome o was present. These action spectra, obtained in CO plus O₂ atmospheres, had maxima only at 432, 550 and 588 nm, attributable to the photodissociation of cytochrome a₃-CO. Even after suppression of cytochrome a₃ activity to 10% of the normal value, no contribution of cytochrome o activity to the photochemical action spectrum was observed. Cytochrome a₃ is therefore the only functional terminal oxidase present in the mitochondria of Crithidia fasciculata.     

Sheet and chain polymeric transition metal complexes formed by N,N-o-phenylenedimethylenebis(pyridin-4-one)

The preparations are reported of the ‘extended reach’ ligand N,N^′-o-phenylene-dimethylenebis(pyridin-4-one) (o-XBP4) and of a range of its metal complexes with Mn(II), Co(II), Ni(II), Cu(II) and Zn(II), two of which have been shown by X-ray studies to have polymeric structures. In the compound [Mn(o-XBP4)(H₂O)₂(NO₃)](NO₃) the o-XBP4 ligands link ‘Mn(H₂O)₂(NO₃)’ units into chains which are then cross-linked into sheets by the bridging action of the coordinated nitrate. In [Cu(o-XBP4)(NO₃)₂] chains are also formed by the bridging action of the o-XBP4 ligands but here they simply pack trough-in-trough with no nitrate cross-linking. X-band EPR spectra are reported for these and the other Mn and Cu compounds as are relevant spectroscopic results for the other complexes.     

猪心线粒体Fo的纯化、重建及其质子转运功能

比较了猪心线粒体F_oF₁-ATPase膜部分F_o的四种纯化方法.结果表明,用NaBr从亚线粒体除去F_oF₁-ATPase的水溶性部分F₁-ATPase后,再以CHAPS增溶,并经蔗糖梯度离心,可获得高纯度的F_o.SDS-聚丙烯酰胺凝胶电泳鉴定表明,纯化的F_o含有b、OSCP（寡霉素敏感授予蛋白）、d、a、e、F₆、IF₁、A6L和c等9种亚基.用去污剂稀释法将纯化的F_o在脂质体上重建后,重建F_o表现较高的被动转运质子活性.这为在体外深入研究F_o的活性、构象与膜脂的关系,以及F_o与F₁-ATPase的组装等提供了很好的实验模型.     

Effect of CuSO4 and Cu(II)(Gly)2 on some indirect assays for superoxide dismutase activity

The effect of CuSO₄ and Cu(II)(Gly)₂ has been compared with that of superoxide dismutase on the ferricytochrome c reduction and on the nitroblue tetrazolium reduction by an enzymic or chemical flux of superoxide anion radicals as well as on o-dianisidine photooxidation. Both CuSO₄ and Cu(II)(Gly)₂ have been found to inhibit ferricytochrome c reduction as well as the aerobic and anaerobic nitroblue tetrazolium reduction with approximately equal efficiency. Unlike superoxide dismutase they proved capable of inhibiting o-dianisdine photooxidation. The effect of copper either as CuSO₄ or as Cu(II)(Cly)₂ has been established as being due to its interference with the indirect assays for superoxide dismutase activity used. The reasons for this interference have been examined and it is concluded that copper can react with a component of the indirect assay system and depending on the method used it either mimics SOD or acts contrary to the enzyme.     

Donut-shaped “miniparticles” in nuclei of human and rat cells

Donut-shaped “miniparticles” were extracted from nuclei of various types of human and rat cells. Electron-microscopic investigations showed these particles were predominantly in sucrose density gradient fractions that had an approximate sedimentation coefficient of 21S. These particles were 113±8_A^o in diameter and had an electron dense center of 29±6_A^o. They appeared to be composed of 8 subunits. Quantitative analysis of the number of these particles by electron-micrographic field counting showed nuclei of tumor samples had a larger amount of the particles than the cytosol. However, normal cell cytosol had a larger number of particles than the nuclei. A group of proteins in the 25, 000–33, 000 molecular weight range was shown to be the main protein component by two dimensional gel electrophoresis.     

Multiple effect of hydroxylamine on mushroom tyrosinase

Mushroom tyrosinase is affected by hydroxylamine (NH₂OH) in several ways. At relatively low concentrations (up to 33 mM) NH₂OH shortens the lag period of tyrosine hydroxylation. The o-dihydroxyphenolase activity of mushroom tyrosinase is slightly stimulated by short exposure to relatively low concentrations ofNH₂OH (1.5 mM). Relatively high concentrations ofNH₂OH (above 20 mM) inhibit the o-dihydroxyphenolase activity of the enzyme and lowers the extent of final pigment production. Preincubation of mushroom tyrosinase with different concentrations ofNH₂OH for different times results in the inactivation of the enzyme. The rate of inactivation occurred much faster under anaerobic than under aerobic conditions. It was also found that NH₂OH changes the spectra of o-quinones prepared chemically or of products formed during the oxidation of o-dihydroxyphenols by mushroom tyrosinase. These spectral changes were attributed to the formation of oximes (mono- or dioximes) as a result of an interaction between o-quinones and NH₂OH. The apparent inhibition exerted by NH₂OH on the o-dihydroxyphenolase activity of mushroom tyrosinase is, in part, due to spectral changes in pigmented product formation and, in part, due to the inactivation of the enzyme by NH₂OH.     

The cytochrome b lysine 329 residue is critical for ubihydroquinone oxidation and proton release at the Qo site of bacterial cytochrome bc1

The ubihydroquinone:cytochrome (cyt) c oxidoreductase (or cyt bc₁) is an important enzyme for photosynthesis and respiration. In bacteria like Rhodobacter capsulatus, this membrane complex has three subunits, the iron?sulfur protein (ISP) with its Fe₂S₂ cluster, cyt c₁ and cyt b, forming two catalytic domains, the Q_o (hydroquinone (QH₂) oxidation) and Q_i (quinone (Q) reduction) sites. At the Q_o site, the electron transfer pathways originating from QH₂ oxidation are known, but their associated proton release routes are less well defined. Earlier, we demonstrated that the His291 of cyt b is important for this latter process. In this work, using the bacterial cyt bc₁ and site directed mutagenesis, we show that Lys329 of cyt b is also critical for electron and proton transfer at the Q_o site. Of the mutants examined, Lys329Arg was photosynthesis proficient and had quasi-wild type cyt bc₁ activity. In contrast, the Lys329Ala and Lys329Asp were photosynthesis-impaired and contained defective but assembled cyt bc₁. In particular, the bifurcated electron transfer and associated proton(s) release reactions occurring during QH₂ oxidation were drastically impaired in Lys329Asp mutant. Furthermore, in silico docking studies showed that in this mutant the location and the H-bonding network around the Fe₂S₂ cluster of ISP on cyt b surface was different than the wild type enzyme. Based on these experimental findings and theoretical considerations, we propose that the presence of a positive charge at position 329 of cyt b is critical for efficient electron transfer and proton release for QH₂ oxidation at the Q_o site of cyt bc₁.     

Discrimination between two possible reaction sequences that create potential risk of generation of deleterious radicals by cytochrome bc1: Implications for the mechanism of superoxide production

In addition to its bioenergetic function of building up proton motive force, cytochrome bc₁ can be a source of superoxide. One-electron reduction of oxygen is believed to occur from semiquinone (SQ_o) formed at the quinone oxidation/reduction Q_o site (Q_o) as a result of single-electron oxidation of quinol by the iron-sulfur cluster (FeS) (semiforward mechanism) or single-electron reduction of quinone by heme b_L (semireverse mechanism). It is hotly debated which mechanism plays a major role in the overall production of superoxide as experimental data supporting either reaction exist. To evaluate a contribution of each of the mechanisms we first measured superoxide production under a broad range of conditions using the mutants of cytochrome bc₁ that severely impeded the oxidation of FeS by cytochrome c₁, changed density of FeS around Q_o by interfering with its movement, or combined these two effects together. We then compared the amount of generated superoxide with mathematical models describing either semiforward or semireverse mechanism framed within a scheme assuming competition between the internal reactions at Q_o and the leakage of electrons on oxygen. We found that only the model of semireverse mechanism correctly reproduced the experimentally measured decrease in ROS for the FeS motion mutants and increase in ROS for the mutants with oxidation of FeS impaired. This strongly suggests that this mechanism dominates in setting steady-state levels of SQ_o that present a risk of generation of superoxide by cytochrome bc₁. Isolation of this reaction sequence from multiplicity of possible reactions at Q_o helps to better understand conditions under which complex III might contribute to ROS generation in vivo.     

Kinetic Variance of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Isolated from Diverse Taxonomic Sources : II. Analysis by Two Dual Label Methods

Two dual label methods were used to investigate kinetic variability of ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (EC 4.1.1.39). In addition to using [1-¹⁴C,5-³H]RuBP (method 1), we describe here the detailed assay with ¹⁴CO₂ and [5-³H]RuBP (method 2), which generates [³H,¹⁴C]3-phosphoglyceric acid and unlabeled (noncontaminating) phosphoglycolate; the carboxylase/oxygenase activity ratio (v_c/v_o) is calculated from ³H/¹⁴C ratios of substrates and products. v_c/v_o was found to be a linear function of [CO₂]/[O₂], constant over a 4-minute assay interval, and invariant of the degree of enzyme activity. Accurately measurable v_c/v_o ratios range from approximately 0.3 to 6. The K_m and V_max of both enzymes may be determined as a composite constant, V_cK_o/V_oK_c. By method 2, the directly compared, relative values at 40 micromolar CO₂ and 1240 micromolar O₂ were: Spinacia oleracea (74), Chlorella pyrenoidosa (31), Plectonema boryanum (32), and Rhodospirillum rubrum (8). With method 1, the values for S. oleracea and R. rubrum were 75, and 9, respectively. Under tight experimental controls, the absolute value for S. oleracea was 69 ± 3.     

The cytochrome bc1 complex inhibitor Ametoctradin has an unusual binding mode

Ametoctradin is an agricultural fungicide that selectively inhibits the cytochrome bc₁ complex of oomycetes. Previous spectrophotometric studies using the purified cytochrome bc₁ complex from Pythium sp. showed that Ametoctradin binds to the Q_o-site of the enzyme. However, as modeling studies suggested a binding mode like that of the substrate ubiquinol, the possibility for a dual Q_o- and Qi-site binding mode was left open.In this work, binding studies and enzyme assays with mitochondrial membrane preparations from Pythium sp. and an S. cerevisiae strain with a modified Q_i-site were used to investigate further the binding mode of Ametoctradin. The results obtained argue that the compound could bind to both the Q_o- and Qi-sites of the cytochrome bc₁ complex and that its position or binding pose in the Q_i-site differs from that of Cyazofamid and Amisulbrom, the two Q_i-site-targeting, anti-oomycetes compounds. Furthermore, the data support the argument that Ametoctradin prefers binding to the reduced cytochrome bc₁ complex. Thus, Ametoctradin has an unusual binding mode and further studies with this compound may offer the opportunity to better understand the catalytic cycle of the cytochrome bc₁ complex.     

Ascochlorin is a novel, specific inhibitor of the mitochondrial cytochrome bc1 complex

Ascochlorin is an isoprenoid antibiotic that is produced by the phytopathogenic fungus Ascochyta viciae. Similar to ascofuranone, which specifically inhibits trypanosome alternative oxidase by acting at the ubiquinol binding domain, ascochlorin is also structurally related to ubiquinol. When added to the mitochondrial preparations isolated from rat liver, or the yeast Pichia (Hansenula) anomala, ascochlorin inhibited the electron transport via CoQ in a fashion comparable to antimycin A and stigmatellin, indicating that this antibiotic acted on the cytochrome bc₁ complex. In contrast to ascochlorin, ascofuranone had much less inhibition on the same activities. On the one hand, like the Q_i site inhibitors antimycin A and funiculosin, ascochlorin induced in H. anomala the expression of nuclear-encoded alternative oxidase gene much more strongly than the Q_o site inhibitors tested. On the other hand, it suppressed the reduction of cytochrome b and the generation of superoxide anion in the presence of antimycin A₃ in a fashion similar to the Q_o site inhibitor myxothiazol. These results suggested that ascochlorin might act at both the Q_i and the Q_o sites of the fungal cytochrome bc₁ complex. Indeed, the altered electron paramagnetic resonance (EPR) lineshape of the Rieske iron-sulfur protein, and the light-induced, time-resolved cytochrome b and c reduction kinetics of Rhodobacter capsulatus cytochrome bc₁ complex in the presence of ascochlorin demonstrated that this inhibitor can bind to both the Q_o and Q_i sites of the bacterial enzyme. Additional experiments using purified bovine cytochrome bc₁ complex showed that ascochlorin inhibits reduction of cytochrome b by ubiquinone through both Q_i and Q_o sites. Moreover, crystal structure of chicken cytochrome bc₁ complex treated with excess ascochlorin revealed clear electron densities that could be attributed to ascochlorin bound at both the Q_i and Q_o sites. Overall findings clearly show that ascochlorin is an unusual cytochrome bc₁ inhibitor that acts at both of the active sites of this enzyme.     

Absence of multiple forms of ribulose 15-bisphosphate carboxylase/oxygenase in mung bean

Ribulose 1,5-bisphosphate carboxylase/oxygenase has been reported to occur in multiple forms in mung bean (Phaseolus aureus) using Sephadex G-200 chromatography. We have isolated this enzyme by identical methodology. The profile from Sephadex G-200 chromatography shows only one peak in contrast to the previous report and we find no evidence to corroborate the conclusions. Where V_c, V_o and K_c, K_o represent V_max and Michaelis constants, respectively, the constant V_cK_o/V_oK_c for the single form is 70 at 40 μM CO₂ and 1200 μM O₂.     

Purification and some properties of a (1→4)-β-d-glucan glucohydrolase associated with the cellulase from the fungus Penicillium funiculosum

The (1→4)-β-d-glucan glucohydrolase from Penicillium funiculosum cellulase was purified to homogeneity by chromatography on DEAE-Sephadex and by iso-electric focusing. The purified component, which had a molecular weight of 65,000 and a pI of 4.65, showed activity on H₃PO₄-swollen cellulose, o-nitrophenyl β-d-glucopyranoside, cellobiose, cellotriose, cellotetraose, and cellopentaose, the K_m values being 172 mg/mL, and 0.77, 10.0, 0.44, 0.77, and 0.37 mm, respectively. d-Glucono-1,5-lactone was a powerful inhibitor of the action of the enzyme on o-nitrophenyl β-d-glucopyranoside (K_i 2.1 μm), cellobiose (K_i 1.95 μm), and cellotriose (K_i 7.9 μm) [cf.d-glucose (K_i 1756 μm)]. On the basis of a Dixon plot, the hydrolysis of o-nitrophenyl β-d-glucopyranoside appeared to be competitively inhibited by d-glucono-1,5-lactone. However, inhibition of hydrolysis by d-glucose was non-competitive, as was that for the gluconolactone-cellobiose and gluconolactone-cellotriose systems. Sophorose, laminaribiose, and gentiobiose were attacked at different rates, but the action on soluble O-(carboxymethyl)cellulose was minimal. The enzyme did not act in synergism with the endo-(1→4)-β-d-glucanase component to solubilise highly ordered cotton cellulose, a behaviour which contrasts with that of the other exo-(1→4)-β-d-glucanase found in the same cellulase, namely, the (1→4)-β-d-glucan cellobiohydrolase.     

X-ray crystallographic investigation and biological activities of Ru(III) complexes containing Schiff base and triphenyl phosphine/arsine

The crystal structures of the complexes [RuCl(Nap-o-phd)(AsPh₃)] and [RuBr(Nap-o-phd)(PPh₃)] (where H₂-Nap-o-phd = N,N′-bis(2-hydroxy-1-naphthaldehyde) o-phenylenediamine) have been determined by single crystal X-ray diffraction techniques. The antibacterial properties of the complexes have also been examined.     

Effect of water activity control on the catalytic performance of surfactant–Arthromyces ramosus peroxidase complex in toluene

Arthromyces ramosus peroxidase (ARP) was successfully modified with a synthetic surfactant for one-electron oxidation reaction of a hydrophobic substrate in toluene. Although UV–visible absorption spectrum of surfactant–ARP complex in toluene showed slight red shift of Soret band compared to that in water, the complex can catalyze oxidation reaction of o-phenylenediamine (o-PDA) with hydrogen peroxide. It appeared that thermodynamic water activity in the reaction system has dominant effect on either the catalytic activity or the stability in the catalytic cycle. Steady-state kinetics under the optimal condition revealed that the specific constant (k_cat/K_m) of ARP complex for o-PDA was 2 orders of magnitude lower than that in aqueous media, while only 13-fold lower for hydrogen peroxide. The reduction of catalytic activity caused by altering the reaction media from water to toluene was found to be mainly due to the low specific constant of ARP complex for o-PDA rather than hydrogen peroxide.     

Interaction of the high-spin Fe atom in the photosystem II reaction center with the quinones QA and QB in purified oxygen-evolving PS II reaction center complex and in PS II particles

EPR signals in the high-spin region were studied at 10 K in photosystem II (PS II) particles and in a purified oxygen-evolving PS II reaction center complex under oxidizing conditions. PS II particles showed EPR peaks at g = 8.0 and 5.6, confirming the recent report by Petrouleas and Diner [(1986) Biochim. Biophys. Acta 849, 264-275]. Addition of 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU) or o-phenanthroline shifted the peaks to be closer to g = 6.0 depending on the medium pH. On the other hand, the PS II reaction center complex showed peaks at g = 6.1 and 7.8, and at g = 6.1 and 6.4, in the absence and presence of o-phenanthroline, respectively. All these peaks were found to be decreased by the illumination at 10 K. These results suggest that the high-spin signals are due to Q₄₀₀, Fe(III) atom interacting with the PS II primary electron acceptor quinone Q_A as reported and that the Fe atom also interacts with the secondary acceptor quinone Q_B. This interaction seems to induce the highly asymmetric ligand coordination of the Fe atom and to be affected by DCMU and o-phenanthroline in a somewhat different manner.     

α and β subunits of F1-ATPase are required for survival of petite mutants in Saccharomyces cerevisiae

Although Saccharomyces cerevisiae can form petite mutants with deletions in mitochondrial DNA (mtDNA) (ρ^?) and can survive complete loss of the organellar genome (ρ^o), the genetic factor(s) that permit(s) survival of ρ^? and ρ^o mutants remain(s) unknown. In this report we show that a function associated with the F₁-ATPase, which is distinct from its role in energy transduction, is required for the petite-positive phenotype of S. cerevisiae. Inactivation of either the α or β subunit, but not the γ, δ, or ? subunit of F₁, renders cells petite-negative. The F₁ complex, or a subcomplex composed of the α and β subunits only, is essential for survival of ρ^o cells and those impaired in electron transport. The activity of F₁ that suppresses ρ^o lethality is independent of the membrane F_o complex, but is associated with an intrinsic ATPase activity. A further demonstration of the ability of F₁ subunits to suppress ρ^o lethality has been achieved by simultaneous expression of S. cerevisiae F₁α and γ subunit genes in Kluyveromyces lactis– which allows this petite-negative yeast to survive the loss of its mtDNA. Consequently, ATP1 and ATP2, in addition to the previously identified AAC2, YME1 and PEL1/PGS1 genes, are required for establishment of ρ^? or ρ^o mutations in S. cerevisiae.