Kondo Insulator to Semimetal Transformation Tuned by Spin-Orbit Coupling.

Recent theoretical studies of topologically nontrivial electronic states in Kondo insulators have pointed to the importance of spin-orbit coupling (SOC) for stabilizing these states. However, systematic experimental studies that tune the SOC parameter λ_{SOC} in Kondo insulators remain elusive. The main reason is that variations of (chemical) pressure or doping strongly influence the Kondo coupling J_{K} and the chemical potential µ-both essential parameters determining the ground state of the material-and thus possible λ_{SOC} tuning effects have remained unnoticed. Here, we present the successful growth of the substitution series Ce_{3}Bi_{4}(Pt_{1-x}Pd_{x})_{3} (0≤x≤1) of the archetypal (noncentrosymmetric) Kondo insulator Ce_{3}Bi_{4}Pt_{3}. The Pt-Pd substitution is isostructural, isoelectronic, and isosize, and it therefore is likely to leave J_{K} and µ essentially unchanged. By contrast, the large mass difference between the 5d element Pt and the 4d element Pd leads to a large difference in λ_{SOC}, which thus is the dominating tuning parameter in the series. Surprisingly, with increasing x (decreasing λ_{SOC}), we observe a Kondo insulator to semimetal transition, demonstrating an unprecedented drastic influence of the SOC. The fully substituted end compound Ce_{3}Bi_{4}Pd_{3} shows thermodynamic signatures of a recently predicted Weyl-Kondo semimetal.

Singular charge fluctuations at a magnetic quantum critical point.

Strange metal behavior is ubiquitous in correlated materials, ranging from cuprate superconductors to bilayer graphene, and may arise from physics beyond the quantum fluctuations of a Landau order parameter. In quantum-critical heavy-fermion antiferromagnets, such physics may be realized as critical Kondo entanglement of spin and charge and probed with optical conductivity. We present terahertz time-domain transmission spectroscopy on molecular beam epitaxy-grown thin films of YbRh2Si2, a model strange-metal compound. We observed frequency over temperature scaling of the optical conductivity as a hallmark of beyond-Landau quantum criticality. Our discovery suggests that critical charge fluctuations play a central role in the strange metal behavior, elucidating one of the long-standing mysteries of correlated quantum matter.

Kondo-like phonon scattering in thermoelectric clathrates.

Crystalline solids are generally known as excellent heat conductors, amorphous materials or glasses as thermal insulators. It has thus come as a surprise that certain crystal structures defy this paradigm. A prominent example are type-I clathrates and other materials with guest-host structures. They sustain low-energy Einstein-like modes in their phonon spectra, but are also prone to various types of disorder and phonon-electron scattering and thus the mechanism responsible for their ultralow thermal conductivities has remained elusive. Our thermodynamic and transport measurements on various clathrate single crystal series and their comparison with ab initio simulations reveal an all phononic Kondo effect as origin. This insight devises design strategies to further suppress the thermal conductivity of clathrates and other related materials classes, with relevance for thermoelectric waste heat recovery and, more generally, phononic applications. It may also trigger theoretical work on strong correlation effects in phonon systems.

The effect of 1-ethyl-3(3-dimethylaminopropyl)carbodiimide on calcium binding and associated changes in chloroplast structure and chlorophyll a fluorescence in spinach chloroplasts.

1. Chemical modification of carboxyl groups on the chloroplast membrane with a water-soluble carbodiimide plus a nucleophile caused inhibition of Ca-2plus binding. 2. Both binding sites were affected and showed a decrease in the number of binding sites and an increase in the dissociation constant. 3. Cation-induced changes in chlorophyll a fluorescence and structural changes (deltaA540) were inhibited at the same carbodiimide concentrations as Ca-2plus binding, emphasizing the relationship between these processes. 4. Chloroplasts that were illuminated with high intensity light for short time periods showed a decrease in the carbodiimide-mediated inhibition of Ca-2plus binding.

Inhibition of cytochrome c oxidase function by dicyclohexylcarbodiimide.

Dicyclohexylcarbodiimide (DCCD) reacted with beef heart cytochrome c oxidase in inhibit the proton-pumping function of this enzyme and to a lesser extent to inhibit electron transfer. The modification of cytochrome c oxidase in detergent dispersion or in vesicular membranes was in subunits II-IV. Labelling followed by fragmentation studies showed that there is one major site of modification in subunit III. DCCD was also incorporated into several sites in subunit II and at least one site of subunit IV. The major site in subunit III has a specificity for DCCD at least one order of magnitude greater than that of other sites (in subunits II and IV). Its modification could account for all of the observed effects of the reagent, at least for low concentrations of DCCD. Labelling of subunit II by DCCD was blocked by prior covalent attachment of arylazidocytochrome c, a cytochrome c derivative which binds to the high-affinity binding site for the substrate. The major site of DCCD binding in subunit III was sequenced. The label was found in glutamic acid 90 which is in a sequence of eight amino acids remarkably similar to the DCCD-binding site within the proteolipid protein of the mitochondrial ATP synthetase.

Homology between bacterial DNA and bovine mitochondrial DNA encoding cytochrome c oxidase subunit III.

A segment of mitochondrial DNA encoding the bovine cytochrome c oxidase subunit III gene was isolated and inserted into an Escherichia coli plasmid vector. A 556 base pair fragment of the insert DNA representing about 70% of the 3'-end of the subunit III gene was used to search for homology with bacterial DNA from strains that contain heme aa3-type cytochrome c oxidases. Bacillus subtilis, Thermus thermophilus, and PS3 DNAs all showed strong hybridization to the probe, whereas Paracoccus denitrificans and Rhodopseudomonas sphaeroides DNAs showed only weak hybridization to the probe, even under low stringency conditions.

Evidence for a conformational change in subunit III of bovine heart mitochondrial cytochrome c oxidase.

The role of subunit III in the function of mitochondrial cytochrome c oxidase is not clearly understood. Previous work has shown that chemical modification of subunit III with N,N'-dicyclohexylcarbodiimide (DCCD) reduced the proton-pumping efficiency of the enzyme by an unknown mechanism. In the current work, we have employed biochemical approaches to determine if a conformational change is occurring within subunit III after DCCD modification. Control and DCCD modified beef heart enzyme were subjected to limited proteolysis in nondenaturing detergent solution. Subunit III in DCCD treated enzyme was more susceptible to chymotrypsin digestion than subunit III in the control enzyme. We also labeled control and DCCD-modified enzyme with iodoacetyl-biotin, a sulfhydryl reagent, and found that subunit III of the DCCD-modified enzyme was more reactive when compared to subunit III of the control enzyme, indicating an increase in reactivity of subunit III upon DCCD binding. The cross linking of subunit III of the enzyme induced by the heterobifunctional reagent, N-succinimidyl(4-azidophenyl -1,3'-dithio)-propionate (SADP), was inhibited by DCCD modification, suggesting that DCCD binding prevents the intersubunit cross linking of subunit III. Our results suggest that DCCD modification of subunit III causes a conformational change, which most likely disrupts critical hydrogen bonds within the subunit and also those at the interface between subunits III and I in the enzyme. The conformational change induced in subunit III by covalent DCCD binding is the most likely mechanism for the previously observed inhibition of proton-pumping activity.

On the synergistic effects of enzymes in food with enzymes in the human body. A literature survey and analytical report.

Recently, a theory has been postulated that suggests that vital enzymes in ingested food interact synergistically with enzymes within the human body and more specifically with enzymes in the digestive tract. Alterations in food enzymes induced by bulk processing including heating and irradiation and also the addition of chemical additives have been proposed to create a decrease in metabolic availability of nutrients, with the long-term consequence being disease. This review of the medical literature provides evidence that enzymes in food do in fact survive during digestion and can indeed, add significantly to the nutritive value of ingested foodstuffs. Examples of enzyme synergy in human nutrition are provided in whole grains, milk and dairy products, beans and seeds, and meat products. A bibliography on this interesting finding is included as well as concluding remarks on enzyme synergy and its putative interaction with cell metabolism. Finally, the interaction of enzyme synergy with disease is discussed.

Effects of food processing on the thermodynamic and nutritive value of foods: literature and database survey.

One of the goals of our society is to provide adequate nourishment for the general population of humans. In the strictness sense, the foodstuffs which we ingest are bundles of thermodynamic energy. In our post-industrial society, food producers provide society with the bioenergetic content of foods, while stabilizing the food in a non-perishable form that enables the consumer to access foods that are convenient and nutritious. As our modern society developed, the processing of foodstuffs increased to allow consumers flexibility in their choice in which foods to eat (based on nutritional content and amount of post-harvest processing). The thermodynamic energy content of foodstuffs is well documented in the literature by the use of bomb calorimetry measurements. Here, we determine the effects of processing (in most cases by the application of heat) on the thermodynamic energy content of foods in order to investigate the role of processing in daily nutritional needs. We also examine which processing procedures affect the nutritive quality (vitamin and mineral content) and critically assess the rational, advantages and disadvantages of additives to food. Finally, we discuss the role of endogenous enzymes in foods not only on the nutritive quality of the food but also on the freshness and flavor of the food. Our results show that a significant decrease in thermodynamic energy content occurs in fruits, vegetables, and meat products upon processing that is independent of water content. No significant change in energy content was observed in cereals, sugars, grains, fats and oils, and nuts. The vitamin content of most foods was most dramatically decreased by canning while smaller effects were observed upon blanching and freezing. We found that most food additives had very little effect on thermodynamic energy content due to their presence in minute quantities and that most were added to preserve the foodstuff or supplement its vitamin content. The endogenous food enzymes while aiding in digestibility of some foods (yogurt or grains) also helped some foods have a more palatable taste. Our conclusions are there is some scientific merit to the idea that enzymes in food can act synergistically with those in the human body to facilitate maximum nutritive value of foods.

Intestinal ischemia after allogeneic stem cell transplantation: a report of four cases.

Gastrointestinal ischemia after allogeneic bone marrow transplantation is a rare complication not well-described in the literature. Herein we retrospectively review charts of four patients who developed intestinal ischemia after allogeneic bone marrow transplantation at our institution. The patients were found to be predominately younger males who presented with nonspecific abdominal pain. Graft-versus-host disease was a common finding among all patients. Laboratory values suggestive of microangiopathy were present in two patients. Obesity and hypertriglyceridemia were cardiovascular risk factors found in these patients. The development of thrombotic microangiopathy and cardiovascular risk factors after allogeneic bone marrow transplantation may predispose patients to gastrointestinal ischemia and may portend a poor prognosis.

Phospholipid vesicles containing bovine heart mitochondrial cytochrome c oxidase exhibit proton translocating activity in the presence of gramicidin.

Phospholipid vesicles containing bovine heart mitochondrial cytochrome c oxidase (COV) were characterized for electron transfer and proton translocating activities in the presence of the mobile potassium ionophore, valinomycin, and the channel-forming ionophore, gramicidin, in order to determine if the ionophores modify the functional properties of the enzyme. In agreement with previous work, incubation of COV with valinomycin resulted in a perturbation of the absorbance spectrum of oxidized heme aa3 in the Soret region (430 nm); gramicidin had no effect on the heme aa3 absorbance spectrum. Different concentrations of the two ionophores were required for maximum respiratory control ratios in COV; 40- to 70-fold higher concentrations of valinomycin were required to completely uncouple electron transfer activity when compared to gramidicin. The proton translocating activity of COV incubated with each inophore gave a similar apparent proton translocated to electron transferred stoichiometry (H+/e- ratio) of 0.66 +/- 0.10. However, COV treated with low concentrations of gramicidin (0.14 mg/g phospholipid) exhibited 1.5- to 2.5-fold higher rates of alkalinization of the extravesicular media after the initial proton translocation reaction than did COV treated with valinomycin, suggesting that gramicidin allows more rapid equilibration of protons across the phospholipid bilayer during the proton translocation assay. Moreover, at higher concentrations of gramicidin (1.4 mg/g phospholipid), the observed H+/e- ratio decreased to 0.280 +/- 0.020, while the rate of alkalinization increased an additional 2-fold, suggesting that at higher concentrations, gramicidin acts as a proton ionophore. These results support the hypothesis that cytochrome c oxidase is a redox-linked proton pump that operates at similar efficiencies in the presence of either ionophore. Low concentrations of gramicidin dissipate the membrane potential in COV most likely by a channel mechanism that is different from the carrier mechanism of valinomycin, yet does not make the phospholipid bilayer freely permeable to protons.

Characterization of electron-transfer and proton-translocation activities in bovine heart mitochondrial cytochrome c oxidase deficient in subunit III.

The electron-transfer and proton-translocation activities of cytochrome c oxidase deficient in subunit III (Mr 29 884) prepared by native gel electrophoresis [Ludwig, B., Downer, N. W., & Capaldi, R. A. (1979) Biochemistry 18, 1401-1407] have been investigated. This preparation has been depleted of 82-87% of its subunit III content as quantitated by Coomassie Brilliant Blue staining intensity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and [14C]dicyclohexylcarbodiimide labeling. The maximum rate of electron transfer of the subunit III deficient enzyme at pH 6.5 is 383 s-1, 78% of control enzyme. Neither the high-affinity site (Km = 10(-8) M) nor the low-affinity site (Km = 10(-6) M) of the cytochrome c kinetic interaction with cytochrome c oxidase is affected by the removal of subunit III. Subunit III deficient cytochrome c oxidase retains the ability to bind cytochrome c in both the high- and low-affinity sites as determined in direct thermodynamic binding experiments. Liposomes containing this preparation exhibit a respiratory control ratio [Hinkle, P. C., Kim, J. J., & Racker, E. (1972) J. Biol. Chem. 247, 1338-1341] of 3.9, while liposomes containing control enzyme exhibit a ratio of 4.3, suggesting that they have a similar proton permeability. Vectorial proton translocation initiated by the addition of ferrocytochrome c in liposomes containing subunit III deficient enzyme is decreased by 64% compared to those containing control enzyme. When the proton-translocated to electron-transferred ratio is measured in these phospholipid vesicles at constant enzyme turnover, removal of subunit III from the enzyme decreases the ratio from 0.52 to 0.21, a 60% decrease.(ABSTRACT TRUNCATED AT 250 WORDS)

On the role of subunit III in proton translocation in cytochrome c oxidase.

Mammalian mitochondrial cytochrome c oxidase catalyzes the transfer of electrons from ferrocytochrome c to molecular oxygen in the respiratory chain, while conserving the energy released during its electron transfer reactions by the vectorial movement of protons across the inner membrane of the mitochondrion. The protein domain that translocates the protons across the membrane is currently unknown. Recent research efforts have investigated the role of one of the transmembrane subunits of the enzyme (III, Mr 29,884) in the vectorial proton translocation reaction. The data that favor subunit III as integral in vectorial proton translocation as well as the data that support a more peripheral role for subunit III in proton translocation are reviewed. Possible experimental approaches to clarify this issue are presented and a general model discussed.

Detection of bovine heart mitochondrial cytochrome c oxidase dimers in Triton X-100 and phospholipid vesicles by chemical cross-linking.

Bovine heart cytochrome c oxidase is a multisubunit enzyme whose oligomeric state is dependent on its detergent or phospholipid environment. We have utilized the cleavable, heterobifunctional cross-linking reagent N-succinimidyl 3-[(4-azidophenyl)dithio]propionate (SADP) to detect cytochrome c oxidase dimers. Monomeric or dimeric enzyme dispersed in Triton X-100 (as assessed by sedimentation velocity measurements) was reacted with SADP. A unique intersubunit cross-link having an apparent molecular mass of 136 kDa was identified in the dimeric enzyme; this product was insensitive to limited proteolysis by trypsin and contained a cross-link between two adjacent monomers. Two-dimensional NaDodSO4-PAGE (the second dimension containing beta-mercaptoethanol to cleave the cross-linking reagent) indicated that subunit I was the major component of the dimer-specific cross-link. The dimer-specific cross-link created by SADP was observed in phospholipid vesicles [cardiolipin/phosphatidylcholine (1:20, w/w)] containing dimeric (2 microM heme aa3) enzyme; a low yield of dimer-specific cross-link was observed in liposomes containing 6 microM (heme aa3) monomeric enzyme. The 136-kDa cross-link was not observed in liposomes containing 2 microM (heme aa3) monomeric enzyme. These results indicate that subunit I from each monomer may provide one site of interaction between monomers in the dimeric form of the enzyme and that cytochrome c oxidase monomers may reassociate to form dimeric complexes in phospholipid vesicles.

Phospholipid vesicles containing bovine heart mitochondrial cytochrome c oxidase and subunit III-deficient enzyme: analysis of respiratory control and proton translocating activities.

Phospholipid vesicles containing bovine heart mitochondrial cytochrome c oxidase (COV) or subunit III (Mr 29884)-deficient enzyme (COV-III) were characterized for electron transfer and proton translocating activities in order to investigate the relationship between the respiratory control ratio (RCR) and the apparent proton translocated to electron transferred stoichiometry (H+/e- ratio) in these preparations. We did not observe a quantitative correlation between the RCR value and the H+/e- ratio in the preparations. Significant deviation between these two parameters was observed in COV-III and also in COV. However, a new parameter, RCRval, did show a linear relationship with the H+/e- ratio of each preparation. Subunit III (SIII)-deficient cytochrome c oxidase isolated by either native gel electrophoresis or chymotrypsin treatment and incorporated into COV-III exhibited H+/e- ratios of 0.34 +/- 0.10, compared to 0.63 +/- 0.09 for COV, emphasizing that the 50% decrease of proton translocating activity is independent of the method of removal of SIII from the enzyme. COV and COV-III also showed similar rates of alkalinization of the extravesicular media after the initial proton translocation reaction (0.07-0.09 neq OH-/s), suggesting that these two preparations had similar endogenous proton permeabilities. In contrast, cytochrome c oxidase (COX) treated with Triton X-100 (3 mg/mg COX) and incorporated into phospholipid vesicles [COV (+TX)] exhibited slower rates of alkalinization (0.04 neq OH-/s), while having a H+/e- ratio similar to that of COV (0.66 +/- 0.10). The passive proton permeabilities of these preparations were tested by valinomycin-induced K+/H+ exchange activity. COV (+TX) and COV-III exhibited similar pseudo-first-order rate constants (10 peq OH-/s), while COV had a 20-fold higher rate constant. These results taken together suggest that the different preparations of COX-containing phospholipid vesicles have different biophysical properties. In addition, the decrease in proton-pumping activity observed in COV-III is due to removal of SIII from COX, suggesting that SIII may act either as a passive proton-conducting channel or as a regulator of COX conformation and/or functional activities.

Characterization of electron transfer and proton translocation activities in trypsin-treated bovine heart mitochondrial cytochrome c oxidase.

Bovine heart mitochondrial cytochrome c oxidase has been treated with trypsin in order to investigate the role of components a, b, and c (nomenclature of Capaldi) in cytochrome c binding, electron transfer, and proton-pumping activities. Cytochrome c oxidase was dispersed in nondenaturing detergent solution (B. Ludwig, N. W. Downer, and R. A. Capaldi (1979) Biochemistry 18, 1401) and treated with trypsin. This treatment inhibited electron transfer activity by 9% when compared to a similarly treated control in a polarographic assay (493 s-1) and had no large effect on the high affinity (Km = 6.1 X 10(-8) M) or low affinity (Km = 2.2 X 10(-6) M) sites of cytochrome c interaction with cytochrome c oxidase. Direct thermodynamic binding experiments with cytochrome c showed that neither the high affinity (1.04 +/- 0.06 mol cytochrome c/mol cytochrome c oxidase) nor the high-plus-low affinity (2.21 +/- 0.15 mol cytochrome c/mol cytochrome c oxidase) binding sites of cytochrome c on the enzyme were perturbed by the trypsin treatment. Control and trypsin-treated enzyme incorporated into phospholipid vesicles (prepared by the cholate dialysis method) exhibited respiratory control ratios of 6.5 +/- 0.7 and 6.3 +/- 0.6, respectively. The vectorial proton translocation activity in the phospholipid vesicles was unaffected by trypsin treatment with proton translocated to electron transferred ratios being equivalent to the control. NaDodSO4-PAGE showed that components a, b, and c were completely removed by the trypsin treatment. [14C]Iodoacetamide labeling experiments showed that the content of component c in the enzyme was depleted by 85% and that greater than 50% of component a was cleaved upon the trypsin treatment. These results suggest that components a, b, and c are not required for maximum electron transfer and proton translocation activities in the isolated enzyme.

Structure of the cytochrome c oxidase complex: labeling by hydrophilic and hydrophobic protein modifying reagents.

Beef heart cytochrome c oxidase has been reacted with [35S]diazobenzenesulfonate ([35S]DABS), [35S]-N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate ([35S]NAP-taurine), and two different radioactive arylazidophospholipids. The labeling of the seven different subunits of the enzyme with these protein modifying reagents has been examined. DABS, a water-soluble, lipid-insoluble reagent, reacted with subunits II, III, IV, V, and VII but labeled I or VI only poorly. The arylazidophospholipids, probes for the bilayer-intercalated portion of cytochrome c oxidase, labeled I, III, and VII heavily and II and IV lightly but did not react with V or VI. NAP-taurine labeled all of the subunits of cytochrome c oxidase. Evidence is presented that this latter reagent reacts with the enzyme from outside the bilayer, and the pattern of labeling with the different hydrophilic and hydrophobic labeling reagents is used to derive a model for the arrangement of subunits in cytochrome c oxidase.

Chemical labeling studies on bovine heart mitochondrial cytochrome c oxidase dispersed in nonionic detergents.

In order to investigate the structural interactions of nonionic detergents with bovine heart mitochondrial cytochrome c oxidase (COX), a series of hydrophilic chemical modification reagents were used to map regions on COX which are not shielded by dodecyl beta-D-maltoside (DM), Triton X-100 (TX-100), and Tween 80 (TW-80). Low levels of incorporation of the chemical reagents [35S]benzenediazoniumsulfonate (DABS) and N-succinimidyl [3H]propionate (SP) into COX dispersed in TW-80 indicate that the bulky headgroup and hydrophobic moiety of this detergent effectively shield the enzyme from the aqueous environment. Subunits II and Va/Vb [nomenclature of Merle, P., & Kadenbach, B. (1982) Eur. J. Biochem. 125, 239-244] show an increased reactivity to [35S]DABS and [3H]SP in TW-80 and may reflect an increased exposure of these subunits to the aqueous phase in comparison to COX dispersed in TX-100 or DM. More [35S]DABS is incorporated into COX in DM than TX-100-dispersed enzyme; DABS heavily labels subunits III, VIa, and VIb in DM. While COX in TX-100 is more reactive with [3H]SP than DM-dispersed enzyme, there is no difference in the distribution of label (either DABS or SP) within the subunits of COX in DM or TX-100. Increased surface exposure of COX in TX-100 is indicated by an enhanced sensitivity of COX electron-transfer activity in enzyme chemically modified by the cross-linking reagent N-succinimidyl 3-[(4-azidophenyl)dithio]propionate (SADP) in TX-100 as compared to enzyme chemically cross-linked in the other detergents.(ABSTRACT TRUNCATED AT 250 WORDS)

High yield purification of a four subunit caa3-type cytochrome oxidase from the thermophilic bacterium Bacillus PS3 using fast protein liquid chromatography.

The thermophilic bacterium, Bacillus PS3, was grown in a vigorously aerated nutrient broth at 65 degrees C with 100 mM glutamic acid serving as a supplemental carbon and nitrogen source. These growth conditions resulted in membranes highly enriched in cytochrome c oxidase (COX) [23.32 +/- 4.32 nmol heme a/g of cells (n = 5)], which is nearly a threefold higher concentration of COX (heme caa3-type) than previously reported for this organism. A new high-yield purification of COX was performed by extracting the bacterial membranes with Triton X-100 (7 mg/mg protein), followed by ion-exchange fast liquid protein chromatography using a QAE (trimethyl ammonium) resin with subsequent hydroxyapatite chromatography and ammonium sulfate fractionation. This purification regime resulted in a 16% yield of cytochrome c oxidase with 20 mg of pure caa3-type COX (13 nmol heme a/mg protein) isolated from 100 g of cells. SDS-PAGE showed that the isolated enzyme had four subunits with apparent Mr of 68, 38, 23, and 13 kDa. In addition, a new 34-kDa peptide was also detected in this preparation, which may represent the ORF1 gene product for this organism. Subunit II (Mr = 38 kDa) of the isolated enzyme was shown to contain covalently bound heme c by using both heme-staining of SDS-PAGE and immunoreactivity with an anti-cytochrome c antibody. The purified enzyme also exhibited high electron transfer activity (340s-1) when assayed at pH 6.5 in the presence of the nonionic detergent, beta-dodecyl maltoside.