Cytochrome oxidase as a proton pump

The general structure of cytochrome oxidase is reviewed and evidence that the enzyme acts as a redox-linked proton pump outlined. The overall H⁺/e ^– stoichiometry of the pump is discussed and results [Wikström (1989),Nature 338, 293] which suggest that only the final two electrons which reduce the peroxide adduct to water are coupled to protein translocated are considered in terms of the restrictions they place on pump mechanisms. Direct and indirect mechanisms for proton translocation are discussed in the context of evidence for redox-linked conformational changes in the enzyme, the role of subunit III, and the nature of the Cu_A site.     

CO2 and water vapour exchange in four alpine herbs at two altitudes and under varying light and temperature conditions

CO₂ and water vapour exchange rates of four alpine herbs namely: Rheum emodi, R. moorcroftianum, Megacarpaea polyandra and Rumex nepalensis were studied under field conditions at 3600 m (natural habitat) and 550 m altitudes. The effect of light and temperature on CO₂ and water vapour exchange was studied in the plants grown at lower altitude. In R. moorcroftianum and R. nepalensis, the average photosynthesis rates were found to be about three times higher at 550 m as compared to that under their natural habitat. However, in M. polyandra, the CO₂ exchange rates were two times higher at 3600 m than at 550 m but in R. emodi, there were virtually no differences at the two altitudes. These results indicate the variations in the CO₂ exchange rates are species specific. The change in growth altitude does not affect this process uniformly.The transpiration rates in R. emodi and M. polyandra were found to be very high at 3600 m compared to 550 m and are attributed to overall higher stomatal conductance in plants of these species, grown at higher altitude. The mid-day closure of stomata and therefore, restriction of transpirational losses of water were observed in all the species at 550 m altitude. In addition to the effect of temperature and relative humidity, the data also indicate some endogenous rhythmic control of stomatal conductance.The temperature optima for photosynthesis was close to 30°C in M. polyandra and around 20°C in the rest of the three species. High temperature and high light intensity, as well as low temperature and high light intensity, adversely affect the net rate of photosynthesis in these species.Both light compensation point and dark respiration rate increased with increasing temperature.The effect of light was more prominent on photosynthesis than the effect of temperature, however, on transpiration the effect of temperature was more prominent than the effect of light intensity.No definite trends were found in stomatal conductance with respect to light and temperature. Generally, the stomatal conductance was highest at 20°C.The study reveals that all these species can easily be cultivated at relatively lower altitudes. However, proper agronomical methodology will need to be developed for better yields.     

1H NMR studies of deuterated ribonuclease HI selectively labeled with protonated amino acids

Summary Two-dimensional (2D)¹H NMR experiments using deuterium labeling have been carried out to investigate the solution structure of ribonuclease HI (RNase HI) fromEscherichia coli (E. coli), which consists of 155 amino acids. To simplify the¹H NMR spectra, two fully deuterated enzymes bearing several prototed amino acids were prepared from an RNase HI overproducing strain ofE. coli grown in an almost fully deuterated medium. One enzyme was selectively labeled by protonated His, He. Val. and Leu. The other was labeled by only protonated His and Ile. The 2D¹H NMR spectra of these deuterated R Nase H1 proteins, selectively labeled with protonated amino acids, were much more simple than those of the normally protonated enzyme. The simplified spectra allowed unambiguous assignments of the resonance peaks and connectivities in COSY and NOESY for the side-chain protons. The spin-lattice relaxation times of the side-chain protons of the buried His residue of the deuterated enzyme became remarkably longer than that of the protonated enzyme. In contrast, the relaxation times of the side-chain protons of exposed His residues remained essentially unchanged.     

Separate,Ca2+-activated K+ and Cl− transport pathways in Ehrlich ascites tumor cells

Summary The net loss of KCl observed in Ehrlich ascites cells during regulatory volume decrease (RVD) following hypotonic exposure involves activation of separate conductive K⁺ and Cl^– transport pathways. RVD is accelerated when a parallel K⁺ transport pathway is provided by addition of gramicidin, indicating that the K⁺ conductance is rate limiting. Addition of ionophore A23187 plus Ca²⁺ also activates separate K⁺ and Cl^– transport pathways, resulting in a hyperpolarization of the cell membrane. A calculation shows that the K⁺ and Cl^– conductance is increased 14-and 10-fold, respectively. Gramicidin fails to accelerate the A23187-induced cell shrinkage, indicating that the Cl^– conductance is rate limiting. An A23187-induced activation of⁴²K and³⁶Cl tracer fluxes is directly demonstrated. RVD and the A23187-induced cell shrinkage both are: (i) inhibited by quinine which blocks the Ca²⁺-activated K⁺ channel. (ii) unaffected by substitution of NO ₃ ^– or SCN^– for Cl^–, and (iii) inhibited by the anti-calmodulin drug pimozide. When the K⁺ channel is blocked by quinine but bypassed by addition of gramicidin, the rate of cell shrinkage can be used to monitor the Cl^– conductance. The Cl^– conductance is increased about 60-fold during RVD. The volume-induced activation of the Cl^– transport pathway is transient, with inactivation within about 10 min. The activation induced by ionophore A23187 in Ca²⁺-free media (probably by release of Ca²⁺ from internal stores) is also transient, whereas the activation is persistent in Ca²⁺-containing media. In the latter case, addition of excess EGTA is followed by inactivation of the Cl^– transport pathway. These findings suggest that a transient increase in free cytosolic Ca²⁺ may account for the transient activation of the Cl^– transport pathway. The activated anion transport pathway is unselective, carrying both Cl^–, Br^–, NO ₃ ^– , and SCN^–. The anti-calmodulin drug pimozide blocks the volume- or A23187-induced Cl^– transport pathway and also blocks the activation of the K⁺ transport pathway. This is demonstrated directly by⁴²K flux experiments and indirectly in media where the dominating anion (SCN^–) has a high ground permeability. A comparison of the A23187-induced K⁺ conductance estimated from⁴²K flux measurements at high external K⁺, and from net K^– flux measurements suggests single-file behavior of the Ca²⁺-activated K⁺ channel. The number of Ca²⁺-activated K⁺ channels is estimated at about 100 per cell.     

Mechanism of inhibition of net ion transport across frog corneal epithelium by calcium channel antagonists

Summary In the isolated bullfrog cornea, three calcium channel antagonists had dose-dependent inhibitory effects on the Cl-originated short-circuit current (SCC). Their order of decreasing potency was bepridil, verapamil and diltiazem. One millimolar diltiazem inhibited the SCC by 98% and subsequent incubation with the calcium ionophore A23187 had no restorative effect. Increasing the bathing solution Ca concentration from 0.05 to 15mm, however, decreased diltiazem's inhibitory efficacy. This antagonist depolarized the intracellular potential differenceV _m from –54 to –18 mV (tear: reference) and the voltage divider ratioFR ₀ decreased from 0.58 to 0.30, suggesting an increase in basolateral membrane electrical resistance. Additional indication of a basolateral membrane effect by the drug was that preincubation with 10⁵ m amphotericin B in Cl-free Ringer's did not eliminate the inhibitory effect of the drug on the Na- and K-elicited SCC. In the absence of amphotericin B in Cl-free Ringer's (SCC=0), 1 ×10³ m diltiazem depolarized theV _m from –78 to –9 mV suggesting that the increase in basolateral membrane resistance was due to K channel blockade. Diltiazem (1×10³ m) significantly decreased cyclic AMP content; however, isoproterenol in the presence of the drug increased cyclic AMP fourfold without having any restorative effect on the inhibited SCC. Therefore, the inhibition of the Cl-originated SCC resulting from an increase in basolateral membrane K resistance is not caused by a decline in cyclic AMP content. In plasma membrane-enriched fractions prepared from broken cell preparations of bovine corneal epithelium, 1×10³ m diltiazem had no inhibitory effects on either Na,K-ATPase or Ca,Mg-ATPase activities. These latter effects further point to the selectivity of diltiazem as an inhibitor of K-channel activity, but do not preclude a Ca-channel blocker effect by the drug in the micromolar range.     

Stability enhancement and circular dichroism spectral anomaly as an indication of non-covalent interactions in histidine- and tyrosine-containing ternary copper(II)—amino acid systems

Visible absorption and CD spectral and potentiometric studies on the His- and Tyr-containing ternary copper(II) complexes Cu(A)(L-B), where A refers to L-His, D-His, or L-Tyr and B to Lys, Tyr, Trp, Phe, Ala, Val, Arg, Glu, Asn, Gln, Ser, or Thr, were made to study ligand-ligand interactions in the complexes. While the CD spectral magnitudes in the d—d region are additive in the absence of side chain interactions and can be estimated from the magnitudes for the ternary systems involving DL-A or DL-B, deviation from the additivity was observed for Cu(L-His)(L-B) (B = LysH, Tyr, Trp, or Phe) and Cu(L-Tyr)(L-Trp). From the stability constants determined at 25 °C and I = 0.1 M (KNO₃), the equilibrium constants, K, for the following hypothetical equilibria were calculated to be large (0.14–0.60) for formation of Cu(L-/D-His)(L-B)(B = Tyr or Trp) and Cu(D-His)(L-Phe) with Cu(en)(L-Ala) as standard: $Cu(A)(L?Ala)+Cu(en)(L?b)\stackrel{K}{?}Cu(A)(L?B)+Cu(en)(L?Ala)$ The positive values indicate the stabilization due to the stacking between the imidazole ring of His and the aromatic side chain of L-B. Solvent dependence of the CD spectra for Cu(L-His)(L-LysH) and Cu(L-His) L-Trp) further supported the existence of the intramolecular electrostatic and hydrophobic interactions.     

Simultaneous and independent effects of abscisic acid on stomata and the photosynthetic apparatus in whole leaves

(±)-Abscisic acid (ABA) at 10^-5 M was added to the transpiration stream of leaves of 16 species (C₃ and C₄, monocotyledons and dicotyledons). Stomatal responses followed one of three patterns: i) stomata that were wide and insensitive to CO₂ initially, closed partially and became sensitive to CO₂; ii) for stomata that were sensitive to CO₂ before the application of ABA, the range of highest sensitivity to CO₂ shifted from high to low intercellular partial pressures of CO₂, for instance in leaves of Zea mays from 170–350 to 70–140 bar; iii) when stomata responded strongly to ABA, their conductance was reduced to a small fraction of the initial conductance, and sensitivity to CO₂ was lost. The photosynthetic apparatus was affected by applications of ABA to various degrees, from no response at all (in agreement with several previous reports on the absence of effects of ABA on photosynthesis) through a temporary decrease of its activity to a lasting reduction. Saturation curves of photosynthesis with respect to the partial pressure of CO₂ in the intercellular spaces indicated that application of ABA could produce three phenomena: i) a reduction of the initial slope of the saturation curve (which indicates a diminished carboxylation efficiency); ii) a reduction of the level of the CO₂-saturated rate of assimilation (which indicates a reduction of the ribulose-1,5-bisphosphate regeneration capacity); and iii) an increase of the CO₂ compensation point. Photosynthesis of isolated mesophyll cells was not affected by ABA treatments. Responses of the stomatal and photosynthetic apparatus were usually synchronous and often proportional to each other, with the result that the partial pressure of CO₂ in the intercellular spaces frequently remained constant in spite of large changes in conductance and assimilation rate. Guard cells and the photosynthetic apparatus were able to recover from effects of ABA applications while the ABA supply continued. Recovery was usually partial, in the case of the photosynthetic apparatus occasionally complete. Abscisic acid did not cause stomatal closure or decreases in the rate of photosynthesis when it was applied during a phase of stomatal opening and induction of photosynthesis that followed a transition from darkness to light.Abbreviations and symbols A rate of CO₂ assimilation - ABA (±)-abscisic acid - c _a partial pressure of CO₂ in the ambient air or in the gas supplied to the leaf chambers - c _i partial pressure of CO₂ in the intercellular spaces of a leaf - e _a partial pressure of H₂O in the air - g conductance for water vapor - J quantum flux - T ₁ leaf temperature     

Stoichiometry of proton uptake in isolated pea chloroplasts under different light intensities

The number of protons released inside the chloroplast thylakoids per electron which is transferred through the electron transport chain (H⁺/e^– ratio) was measured in isolated pea chloroplasts at pH 6.0 under continuous illumination and with methyl viologen as an electron acceptor. At saturating light intensity (200 W · m^–2) (strong light) the H⁺/e^– ratio was 3. At low intensity (0.9 W · m^–2) (weak light) the H⁺/e^– ratio was 2 with dark-adapted chloroplasts, but it was close to 3 with chloroplasts that were preilluminated with strong light. It is shown that the presence of azide in the reaction mixture leads to errors in the determination of the H⁺/e^– ratio due to underestimation of the initial rate of H⁺ efflux on switching off the light. To explain the above data, we assume that transformation of the electron transport chain occurs during illumination with strong light, namely, the Q cycle becomes operative.     

1H NMR study of the interaction of bacteriophage λ Cro protein with the O R 3 operator

The 17 base pair operator O _R ³ oligonucleotide, which is the preferential binding site for the Cro repressor of phage , was studied by two-dimensional NMR spectroscopy. A sequential assignment procedure based on two-dimensional Nuclear Overhauser Effect (NOESY) and scalar coupling correlated (COSY) NMR spectroscopy, together with the knowledge of the oligodesoxynucleotide sequence, made it possible to assign the non-exhangeable base protons and the H1 and the H2-H2 sugar protons of the O _R ³ operator DNA. The pattern of the observed NOE connectivities is consistent with a right-handed helical DNA structure. The base and sugar proton assignments provide the necessary information for further studies of the O _R ³ operator — Cro repressor interaction.Abbreviations COSY correlated spectroscopy - FID free induction decay - NOE nuclear Overhauser effect - NOESY nuclear Overhauser effect spectroscopy - RD relaxation delay - TSP sodium 3-trimethylsilyl-(2,2,3,3-²H₄)propionate - EDTA sodium ethylendiamine tetraacetate     

Stomatal response to air humidity and its relation to stomatal density in a wide range of warm climate species

The gas exchange of 19 widely different warm climate species was observed at different leaf to air vapour pressure deficits (VPD). In all species stomata tended to close as VPD increased resulting in a decrease in net photosynthesis. The absolute reduction in leaf conductance per unit increase in VPD was greatest in those species which had a large leaf conductance at low VPDs. This would be expected even if stomata of all species were equally sensitive. However the percentage reduction in net photosynthesis (used as a measure of the relative sensitivity of stomata of the different species) was also closely related to the maximal conductance at low VPD. Similarily the relative sensitivity of stomata to changes in VPD was closely related to the weighted stomatal density or crowding index.The hypothesis is presented that stomatal closure at different VPDs is related to peristomatal evaporation coupled with a high resistance between the epidermis and the mesophyll and low resistance between the stomatal apparatus and the epidermal cells. This hypothesis is consistent with the greater relative sensitivity of stomata on leaves with a high crowding index.The results and the hypothesis are discussed in the light of selection, for optimal productivity under differing conditions of relative humidity and soil water availablility, by observation of stomatal density and distribution on the two sides of the leaf.Visiting scientist, plant physiologist and research assitant of the Cassava Program