Theoretical study of the H + HCN → H + HNC process

We present a theoretical study on the detailed mechanism and kinetics of the H+HCN →H+HNC process. The potential energy surface was calculated at the complete basis set quantum chemical method, CBS-QB3. The vibrational frequencies and geometries for four isomers (H₂CN, cis-HCNH, trans-HCNH, CNH₂), and seven saddle points (TSn where n = 1 ? 7) are very important and must be considered during the process of formation of the HNC in the reaction were calculated at the B3LYP/6-311G(2d,d,p) level, within CBS-QB3 method. Three different pathways (PW1, PW2, and PW3) were analyzed and the results from the potential energy surface calculations were used to solve the master equation. The results were employed to calculate the thermal rate constant and pathways branching ratio of the title reaction over the temperature range of 300 up to 3000 K. The rate constants for reaction H + HCN → H + HNC were fitted by the modified Arrhenius expressions. Our calculations indicate that the formation of the HNC preferentially occurs via formation of cis–HCNH, the fitted expression is k _{P W2}(T) = 9.98 × 10^?22 T ^2.41 exp(?7.62 kcal.mol^?1/R T) while the predicted overall rate constant k _{O v e r a l l} (T) = 9.45 × 10^?21 T ^2.15 exp(?8.56 kcal.mol^?1/R T) in cm ³ molecule ^?1 s ^?1.

Open image in new window

Graphical Abstract (a) Potential energy surface, (b) thermal rate constants as a function of temperature and (c) the branching ratios (%) of PW1, PW2, PW3 pathways involved in rm H + HCN → H + HNC process.

     

Relation between electron exchange and nuclear vibrations in the H 2 + +H 2 + → H 3 + +p exothermic ion-molecular reaction

The effective cross section for the H ₂ ⁺ +H ₂ ⁺ → H ₃ ⁺ +p reaction in the energy range 5.7–11.5 eV is measured by the split beam method. The maximum of the cross section at an energy of ～8 eV is related to the production of the H ₄ ⁺⁺ compound system. The reaction threshold W _thr≈5 eV provides evidence in favor of the classical model of the H ₂ ⁺ ion with the charge fixed on one of the nuclei throughout the collision event.     

Vector correlations in the F + HO → HF + O reaction and its isotopic variant

The F + H(D)O → HF(DF) + O reactions have been studied using quasi-classical trajectory (QCT) calculation method, based on the three different potential energy surfaces (PESs) of Gomez-Carrasco et al. (J Chem Phys 2004, 121:4605; J Chem Phys 2005, 123:114310; Chem Phys Lett 2007, 435:188). Facilitated with the analysis of the QCT results, the pictures for product scattering and product polarizations have been presented to investigate the vector correlations in the two reactions, with effects of isotope substitution and electronic state as well as collision energy being revealed at a chemical stereodynamical level.     

Proton block of the CLC-5 Cl?/H+ exchanger

CLC-5 is a H⁺/Cl⁻ exchanger that is expressed primarily in endosomes but can traffic to the plasma membrane in overexpression systems. Mutations altering the expression or function of CLC-5 lead to Dent’s disease. Currents mediated by this transporter show extreme outward rectification and are inhibited by acidic extracellular pH. The mechanistic origins of both phenomena are currently not well understood. It has been proposed that rectification arises from the voltage dependence of a H⁺ transport step, and that inhibition of CLC-5 currents by low extracellular pH is a result of a reduction in the driving force for exchange caused by a pH gradient. We show here that the pH dependence of CLC-5 currents arises from H⁺ binding to a single site located halfway through the transmembrane electric field and driving the transport cycle in a less permissive direction, rather than a reduction in the driving force. We propose that protons bind to the extracellular gating glutamate E211 in CLC-5. It has been shown that CLC-5 becomes severely uncoupled when SCN⁻ is the main charge carrier: H⁺ transport is drastically reduced while the rate of anion movement is increased. We found that in these conditions, rectification and pH dependence are unaltered. This implies that H⁺ translocation is not the main cause of rectification. We propose a simple transport cycle model that qualitatively accounts for these findings.     

Long α helices projecting from the membrane as the dimer interface in the voltage-gated H+ channel

The voltage-gated H⁺ channel (Hv) is a H⁺-permeable voltage-sensor domain (VSD) protein that consists of four transmembrane segments (S1–S4). Hv assembles as a dimeric channel and two transmembrane channel domains function cooperatively, which is mediated by the coiled-coil assembly domain in the cytoplasmic C terminus. However, the structural basis of the interdomain interactions remains unknown. Here, we provide a picture of the dimer configuration based on the analyses of interactions among two VSDs and a coiled-coil domain. Systematic mutations of the linker region between S4 of VSD and the coiled-coil showed that the channel gating was altered in the helical periodicity with the linker length, suggesting that two domains are linked by helices. Cross-linking analyses revealed that the two S4 helices were situated closely in the dimeric channel. The interaction interface between the two S4 and the assembly interface of the coiled-coil domain were aligned in the same direction based on the phase angle calculation along α helices. Collectively, we propose that continuous helices stretching from the transmembrane to the cytoplasmic region in the dimeric interface regulate the channel activation in the Hv dimer.     

Do H+ ions obscure electrogenic Na+ and K+ binding in the E1 state of the Na,K-ATPase?

In contrast to other P-type ATPases, the Na,K-ATPase binding and release of ions on the cytoplasmic side, to the state called E1, is not electrogenic with the exception of the third Na+. Since the high-resolution structure of the closely related SR Ca-ATPase in state E1 revealed the ion-binding sites deep inside the transmembrane part of the protein, the missing electrogenicity in state E1 can be explained by an obscuring counter-movement of H+ ions. Evidence for such a mechanism is presented by analysis of pH effects on Na+ and K+ binding and by electrogenic H+ movements in the E1 conformation of the Na,K-ATPase.     

The soybean F3′H protein is localized to the tonoplast in the seed coat hilum

We previously isolated a soybean (Glycine max (L.) Merr.) flavonoid 3'-hydroxylase (F3'H) gene (sf3'h1) corresponding to the T locus, which controls pubescence and seed coat color, from two near-isogenic lines (NILs), To7B (TT) and To7G (tt). The T allele is also associated with chilling tolerance. Here, Western-blot analysis shows that the sf3'h1 protein was predominantly detected in the hilum and funiculus of the immature seed coat in To7B, whereas sf3'h1 was not detected in To7G. A truncated sf3'h1 protein isolated from To7G was detected only upon enrichment by immunoprecipitation. An analysis using diphenylboric acid 2-aminoethyl ester (DBPA) staining revealed that flavonoids accumulated in the hilum and the funiculus in both To7B and To7G. Further, the scavenging activity of the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical in methanol extracts from the funiculus and hilum of To7B was higher than that of To7G. Moreover, the enzymatic activity of F3'H was detected using microsomal fractions from yeast transformed with sf3'h1 from To7B, but not from To7G. These results indicate that sf3'h1 is involved in flavonoid biosynthesis in the seed coat and affects the antioxidant properties of those tissues. As shown by immunofluorescence microscopy, the sf3'h1 protein was detected primarily around the vacuole in the parenchymatic cells of the hilum in To7B. Further immunoelectron microscopy detected sf3'h1 protein on the membranous structure of the vacuole. Based on these observations, we conclude that F3'H, which is a cytochrome P450 monooxygenase and has been found to be localized to the ER in other plant systems, is localized in the tonoplast in soybean.     

How Does the Plant Plasma Membrane H+-ATPase Pump Protons?

The plasma membrane H⁺-ATPase couples ATP hydrolysis to protontransport thereby establishing the driving force for solutetransport into and out of plant cells. As such, this enzymeparticipates in a number of cellular processes important tothe overall physiology of plants. From biochemical studies andthe recent application of molecular approaches, the enzyme reactionmechanism and structure of this protein have been characterized.However, our basic understanding of how this enzyme links theendergonic reaction of ATP hydrolysis to proton translocationis far from complete. In this review, several significant questionsregarding the energy coupling mechanism will be addressed interms of information available on the plant plasma membraneH⁺-ATPase and from studies on other P-type transport ATPases.These questions focus on the chemical nature of proton translocation,how this is linked or driven by the ATP hydrolysis reactionand what role, if any, K⁺ has in the transport process. Key words: Energy coupling, membranes, bioenergetics, ion transport, P-type transport ATPase     

Stimulation of H+transport activity of vacuolar H+ATPase by activation of H+PPase in Kalanchoë blossfeldiana

In Kalanchoë blossfeldiana cv. Tom Thumb the initial rate of ATP-dependent H+-transport into tonoplast vesicles was stimulated up to three times if the H+-ATPase (EC 3.6.1.3) was energized a few minutes after pre-energization of the H+-PPase (EC 3.6.1.1). H+-PPase-activated ATP-dependent H+-transport was observed in plants of K. blossfeldiana cultivated in short day (SD) or long day (LD) conditions expressing different degrees of crassulacean acid metabolism (CAM). However, based on the higher activity and protein amount of H+-PPase and H+-ATPase present in the vacuolar membrane of SD plants the maximum H+-transport activity in the stimulated mode of the H+-ATPase was significantly higher in tonoplast vesicles of SD plants than of LD plants. Hence, a co-ordinated action of the H+-PPase and H+-ATPase at the tonoplast of Kalanchoë could allow a higher transport capacity at the vacuolar membrane when plants perform high CAM. Immunoprecipitation experiments with an antiserum raised against the A-subunit of the vacuolar H+-ATPase of Mesembryanthemum crystallinum L. showed that in SD and LD plants of K. blossfeldiana the H+-PPase was co-precipitated with the vacuolar H+-ATPase holoenzyme. The co-percipitation of the two transport proteins indicates a close structural localization of the H+-PPase and the A-subunit of the vacuolar H+-ATPase.     

pH-dependent regulation of the α-subunit of H+-K+-ATPase (HKα2)

The H(+)-K(+)-ATPase α-subunit (HKα(2)) participates importantly in systemic acid-base homeostasis and defends against metabolic acidosis. We have previously shown that HKα(2) plasma membrane expression is regulated by PKA (Codina J, Liu J, Bleyer AJ, Penn RB, DuBose TD Jr. J Am Soc Nephrol 17: 1833-1840, 2006) and in a separate study demonstrated that genetic ablation of the proton-sensing G(s)-coupled receptor GPR4 results in spontaneous metabolic acidosis (Sun X, Yang LV, Tiegs BC, Arend LJ, McGraw DW, Penn RB, Petrovic S. J Am Soc Nephrol 21: 1745-1755, 2010). In the present study, we investigated the ability of chronic acidosis and GPR4 to regulate HKα(2) expression in HEK-293 cells. Chronic acidosis was modeled in vitro by using multiple methods: reducing media pH by adjusting bicarbonate concentration, adding HCl, or by increasing the ambient concentration of CO(2). PKA activity and HKα(2) protein were monitored by immunoblot analysis, and HKα(2) mRNA, by real-time PCR. Chronic acidosis did not alter the expression of HKα(2) mRNA; however, PKA activity and HKα(2) protein abundance increased when media pH decreased from 7.4 to 6.8. Furthermore, this increase was independent of the method used to create chronic acidosis. Heterologous expression of GPR4 was sufficient to increase both basal and acid-stimulated PKA activity and similarly increase basal and acid-stimulated HKα(2) expression. Collectively, these results suggest that chronic acidosis and GPR4 increase HKα(2) protein by increasing PKA activity without altering HKα(2) mRNA abundance, implicating a regulatory role of pH-activated GPR4 in homeostatic regulation of HKα(2) and acid-base balance.     

Vacuolar H+/Ca2+ transport: who's directing the traffic?

Physiological studies have established the role of plant high-capacity vacuolar H+/Ca2+ exchange activity in ion homeostasis and signal transduction. The molecular characterization and structure-function analyses of these transporters are just beginning to emerge. In yeast, Ca2+ signaling molecules regulate vacuolar H+/Ca2+ exchange. Recently, some of the Ca2+ dependent "molecular relay" molecules have been characterized in plants; however, the regulation of plant vacuolar H+/Ca2+ exchange remains an open question.     

Fusicoccin Binding to Its Plasma Membrane Receptor and the Activation of the Plasma Membrane H+-ATPase (III. Is There a Direct Interaction between the Fusicoccin Receptor and the Plasma Membrane H+-ATPase?)

A radioimmunoassay using antibodies raised against bovine serum albumin-conjugated fusicoccin (FC) was applied to measure FC bound to the plasma membrane (PM) isolated from seedlings of radish (Raphanus sativus L.) and of Arabidopsis thaliana treated in vivo plus or minus the toxin. FC bound to the PM from seedlings treated with 5 [mu]M FC was 2-fold (radish) to 7-fold (A. thaliana) higher than the binding capacity of control PM. FC binding depended on the duration of the in vivo treatment but was unaffected by cycloheximide. When FC binding and the PM H+-ATPase activity were compared under different conditions (in vivo or in vitro treatment of different lengths or with different concentrations of FC), a strict linear relation between FC binding and the activation of the PM H+-ATPase was observed in both plant materials under all the conditions tested. Comparison between the maximum binding capacity and the amount of H+-ATPase observed in PM from the two plant materials suggest a one-to-one stoichiometry between the FC receptor and the PM H+-ATPase.     

Relationship between the F0F1-ATPase and the K+-transport system within the membrane of anaerobically grownEscherichia coli. N,N′-dicyclohexylcarbodiimide-sensitive ATPase activity in mutants with defects in K+-transport

A considerable (2-fold) stimulation of the DCCD-sensitive ATPase activity by K⁺ or Rb⁺, but not by Na⁺, over the range of zero to 100mM was shown in the isolated membranes ofE. coli grown anaerobically in the presence of glucose. This effect was observed only in parent and in thetrkG, but not in thetrkA, trkE, ortrkH mutants. ThetrkG or thetrkH mutant with anunc deletion had a residual ATPase activity not sensitive to DCCD. A stimulation of the DCCD-sensitive ATPase activity by K⁺ was absent in the membranes from bacteria grown anaerobically in the presence of sodium nitrate. Growth of thetrkG, but not of othertrk mutants, in the medium with moderate K⁺ activity did not depend on K⁺ concentration. Under upshock, K⁺ accumulation was essentially higher in thetrkG mutant than in the othertrk mutant. The K⁺-stimulated DCCD-sensitive ATPase activity in the membranes isolated from anaerobically grownE. coli has been shown to depend absolutely on both the F₀F₁ and theTrk system and can be explained by a direct interaction between these transport systems within the membrane of anaerobically grown bacteria with the formation of a single supercomplex functioning as a H⁺-K⁺ pump. ThetrkG gene is most probably not functional in anaerobically grown bacteria.This study was performed at the Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, Illinois 60637.     

Ontogeny of the Na+−H+ exchanger in rat ileal brush-border membrane vesicles

Summary The developmental maturation of Na⁺–H⁺ antiporter was determined using a well-validated brush-border membrane vesicles (BBMV's) technique. Na⁺ uptake represented transport into an osmotically sensitive intravesicular space as evidenced by an osmolality study at equilibrium. An outwardly directed pH gradient (pH inside/pH outside=5.2/7.5) significantly stimulated Na⁺ uptake compared with no pH gradient conditions at all age groups; however, the magnitude of stimulation was significantly different between the age groups. Moreover, the imposition of greater pH gradient across the vesicles resulted in marked stimulation of Na⁺ uptake which increased with advancing age. Na⁺ uptake represented an electroneutral process.The amiloride sensitivity of the pH-stimulated Na⁺ uptake was investigated using [amiloride] 10^–2–10^–5 m. At 10^–3 m amiloride concentration, Na⁺ uptake under pH gradient conditions was inhibited 80, 45, and 20% in BBMV's of adolescent, weanling and suckling rats, respectively. Kinetic studies revealed aK _m for amiloride-sensitive Na⁺ uptake of 21.8±6.4, 24.9±10.9 and 11.8±4.17mm andV _max of 8.76±1.21, 5.38±1.16 and 1.99±0.28 nmol/mg protein/5 sec in adolescent, weanling and suckling rats, respectively. The rate of pH dissipation, as determined by the fluorescence quenching of acridine orange, was similar across membrane preparation of all age groups studied. These findings suggest for the first time the presence of an ileal brush-border membrane Na⁺–H⁺ antiporter system in all ages studied. This system exhibits changes in regard to amiloride sensitivity and kinetic parameters.     

柠条锦鸡儿F3H基因克隆及功能分析

柠条锦鸡儿为豆科灌木,对各种环境胁迫具有较强的适应能力,类黄酮是天然的抗氧化剂,花青素属类黄酮化合物,逆境胁迫会影响植物体内花青素的合成,而黄烷酮3-羟化酶(F3H)是花青素生物合成所必需的一种关键酶。该研究成功分离克隆了柠条锦鸡儿的F3H基因,命名为CkF3H。CkF3H基因的开放阅读框(ORF)为1095 bp,编码364个氨基酸,推测的蛋白质分子量为41.3 kDa,理论等电点为5.9。生物信息学分析发现,CkF3H基因序列与其它植物F3H有较高的一致性,推测CkF3H蛋白与其它植物F3H蛋白具有相似的功能。利用染色体步移法克隆得到CkF3H起始密码子ATG上游468 bp的启动子序列,PlantCARE软件分析表明,该序列具有启动子的基本元件CAAT-box和TATA-box以及多种与逆境胁迫相关的顺式调控元件。实时荧光定量PCR分析表明,CkF3H在柠条的根、茎和叶中均有表达,没有组织特异性;CkF3H的表达受低温、高盐、干旱和高温胁迫的诱导,并且在低温胁迫下,CkF3H的表达还受到光周期的影响。综上所述,研究结果表明CkF3H基因在柠条锦鸡儿适应低温、高盐、干旱和高温胁迫的过程中发挥作用。