Proton electrochemical gradient and phosphate potential in submitochondrial particles

1. The aerobic uptake of inorganic ions, such as ⁸⁶Rb⁺ or ¹²⁵I^?, by submitochondrial particles, is about one order of magnitude lower than the uptake of organic ions, such as acridines or 8-anilino-1-naphthalene sulphonate. The values of ΔpH, the transmembrane pH differential, and Δψ, the transmembrane membrane potential are between 60 and 100 mV when calculated on the inorganic ions and between 150 and 240 mV when calculated on the organic ions. The discrepancy between the ΔpH and Δψ values from organic and inorganic ions is large at high but not at low ion/protein ratios.2. In the absence of weak bases and strong acids the values of $\text{Δ}\text{}\text{?}\text{gm}{}_{\mathrm{<mtext>H</mtext>}}$, the proton electrochemical potential difference, are close to 100 mV and the magnitude of ΔpH and Δψ are similar. Weak bases decrease ΔpH and enhance Δψ. Strong acids decrease Δψ and enhance ΔpH. Interchangeability of ΔpH with Δψ occurs at low concentrations of weak bases and strong acids. High concentrations of weak bases and strong acids cause depression of $\text{Δ}\text{}\text{?}\text{gm}{}_{\mathrm{<mtext>H</mtext>}}$.3. Concentrations of weak bases capable of abolishing ΔpH, do not affect ATP synthesis. Concentrations of strong acids capable of abolishing Δψ affect only slightly ATP synthesis. Concentrations of weak bases and strong acids capable of causing a decline of ΔpH + Δψ inhibit ATP synthesis.4. Depression of $\text{Δ}\text{}\text{?}\text{gm}{}_{\mathrm{<mtext>H</mtext>}}$ is paralleled by inhibition of ATP synthesis and decline of ΔGp, the phosphate potential. Abolition of ATP synthesis occurs only when $\text{Δ}\text{}\text{?}\text{gm}{}_{\mathrm{<mtext>H</mtext>}}$ is below 20 mV. The $\text{ΔG}\text{p}\text{/Δ}\text{}\text{?}\text{gm}{}_{\mathrm{<mtext>H</mtext>}}$ ratio increases hyperbolically with the decrease of $\text{Δ}\text{}\text{?}\text{gm}{}_{\mathrm{<mtext>H</mtext>}}$.     

Flash-induced photophosphorylation in chloroplasts with activated ATPase

Chloroplasts which were rapidly isolated from illuminated leaves showed activity of ATP hydrolysis at a level much higher than that of the dark control. Under the high-intensity illumination or under repetitive flash excitation, the activated chloroplasts synthesized more ATP than those with a low ATP hydrolysis activity. formed under repetitive flashes was smaller in the activated chloroplasts than in the inactive chloroplasts. The inhibition of ATP yield per flash by valinomycin or nigericin in the presence of K⁺ was stronger in the inactive chloroplasts than in the activated chloroplast. ATP synthesis in the activated chloroplasts seems to have a lower threshold.     

Localized coupling in oxidative phosphorylation by mitochondria from Jerusalem artichoke (Helianthus tuberosus)

Delocalized chemiosmotic coupling of oxidative phosphorylation requires that a single-value correlation exists between the extent of and the kinetic parameters of respiration and ATP synthesis. This expectation was tested experimentally in nigericin-treated plant mitochondria in single combined experiments, in which simultaneously respiration (in State 3 and in State 4) was measured polarographically, FΔψ (which under these conditions was equivalent to ) was evaluated potentiometrically from the uptake of tetraphenylphosphonium⁺ and the rate of phosphorylation was estimated from the transient depolarization of mitochondria during State 4-State 3-State 4 transitions. The steady-state rates of the different biochemical reactions were progressively inhibited by specific inhibitors active with different modalities on various steps of the energy-transducing process: succinate respiration was inhibited competitively with malonate or noncompetitively with antimycin A, or by limiting the rate of transport into the mitochondria of the respiratory substrate with phenylsuccinate; was dissipated by uncoupling with increasing concentrations of valinomycin; ADP phosphorylation was limited with oligomycin. The results indicate generally that when the rate of respiratory electron flow is decreased, a parallel inhibition of the rate of phosphorylation is also observed, while very limited effects can be detected on the extent of . This behavior is in marked contrast to the effect of uncoupling where the decreased rate of ATP synthesis is clearly due to energy limitation. Extending previous observations in bacterial photosynthesis and in respiration by animal mitochondria and submitochondrial particles the results indicate, therefore, that respiration tightly controls the rate of ATP synthesis, with a mechanism largely independent of . These data cannot be reconciled with a delocalized chemiosmotic coupling model.     

Activation-deactivation reactions in the ATPase enzyme in Rhodospirillum rubrum chromatophores

(1) The ATPase enzyme in untreated chromatophores from Rhodospirillum rubrum is in a low-activity state (designated as E°). It can be activated by application of a transmembrane generated by light-induced electron transport, or by application of acid-base jumps. (2) After rapid dissipation of the light-induced , the active state of the ATPase enzyme decays (in the absence of added substrates or products) to a low-activity state (designated as E′), with a half-time of the order of 2–4 min. This state differs from E° in that E′ (but not E°) can be rapidly reactivated by addition of substrate, but only when the Mg²⁺ concentration is kept below 20–30 μM. Since this is characteristic of an activated enzyme containing tightly bound ADP (Slooten, L. and Nuyten, A. (1981) Biochim. Biophys. Acta 638, 313–326), it is suggested that release of endogenous, tightly bound ADP is one of the factors involved in activation of the ATPase enzyme.     

H+ transport in the evolution of photosynthesis

Extant photosynthetic organisms all appear to use transmembrane H⁺ fluxes as the coupling agent in the use of light energy in ATP synthesis. In the steady-state there is a large H⁺ free energy difference across the coupling membrane, and when this is reflected as a light-induced change in pH of the phase (cytosol or stroma) containing the enzymes of carbon assimilation, the H⁺ transport can have an informational role in activating and inactivating enzymes.The earliest organisms probably lived fermentatively (substrate-level phosphorylation) in an anaerobic environment provided with organic solutes synthesised abiotically. There are good reasons for believing that one of the earliest primary active transport systems (interconverting chemical and electrical/osmotic energy) was an H⁺ extrusion pump powered by ATP or PP_i. Its initial function was extrusion of excess H⁺ from the fermenting cells, and the support of a number of co-transport processes. The earliest energetic use of light energy is envisaged as being the energization of an alternative H⁺ extrusion pump, with bacteriorhodopsin or (bacterio-) chlorophyll as the pigment. The former type of cyclic photoredox system (Halobacterium-type) is simpler than the latter: a “pre-respiratory” chemical redox H⁺ pump may have preceded the (bacterio-) chlorophyll-based process. Any of these H⁺ pumps could spare the use of fermentative ATP in powering active H⁺ efflux and would thus have been favoured as fermentative substrates became scarce; eventually the larger generated by the light-powered H⁺ pump was used to drive the ATP-powered H⁺ pump backwards and thus generate ATP with light as the ultimate energy source.Scarcity of suitable reductants for biosynthesis as life proliferated provided a selective impetus for a non-cyclic photoredox system which could use light energy to generate a low-potential reductant at the expense of more readily available higher-potential reductants. The non-cyclic photoredox system is not possible in its simplest form (with all the redox energy coming from excitation energy of one or more photoreactions) in the bacteriorhodopsin line of evolution. Such a simple photoredox system is found in the Chlorobiaceae; even if (as seems likely) the non-cyclic photoredox process generates a (and thus, potentially, ATP), some of the ATP needed for CO₂ fixation and cell growth must be generated by a cyclic photoredox system.In the extant purple bacteria the generation of low-potential reductant involves a non-cyclic photoredox pathway which produces a reductant unable to reduce NAD⁺; the “energy gap” is spanned by “reverse electron transfer” which uses energy from a . It is not clear if this energetic requirement for the H⁺ gradient can be quantitatively satisfied from a non-cyclic photoredox H⁺ transport; it is certain that there is a major requirement for cyclic photoredox H⁺ pumping in these organisms.The photosynthetic bacteria are today restricted to reducing (low E_h) environments similar to those found in the early, anoxic earth; they are unable to use very weak reductants as donors for non-cyclic photoredox processes. As the sources of even weakly reducing donors (other than H₂O) on the primitive earth were depleted the two photoreactions scheme of extant O₂-producers evolved by modification of the bacterial photoreaction. This non-cyclic photoredox process is definitely H⁺-translocating and the role of cyclic photoredox processes in ATP generation in O₂-evolvers is smaller than in photosynthetic bacteria.In parallel with the biochemical and biophysical changes in the photosystems there was a morphological evolution, with an increasing tendency for “internalisation” of the photoredox processes (originally present in the plasma membrane, as in extant Chlorobineae) into thylakoids (as in most Rhodospirillineae, Cyanobacteria and in all eukaryotes). With a plasmalemma-located photoredox system, and the constraints of a fixed, alkaline external pH and the cytoplasmic pH of 7–8, the would be generated largely as an electrical P.D. The presence of a phase (intrathylakoid space) with a “negotiable pH” would permit the generation and use of a largely present as a pH gradient.In both cases illumination can cause an increase in cytoplasmic (stromal) pH over the dark value; this is an important aspect of the regulation of “phototrophic” and “heterotrophic” enzyme systems in the light and in the dark. However, it is argued that these differences in pH are not absolutely light-dependent unless they depend upon some more uniquely light-dependent signal, probably based on a redox component only generated in the light.     

Enthalpy and volume changes accompanying electron transfer from P-870 to quinones in Rhodopseudomonas sphaeroides reaction centers

A capacitor microphone was used to measure the enthalpy and volume changes that accompany the electron transfer reactions, $\text{PQ}{}_{\mathrm{A}}\text{→}\text{hv}\text{P}{}^{\mathrm{+}}\text{Q}{}^{\mathrm{?}}{}_{\mathrm{A}}\text{and PQ}{}_{\mathrm{A}}\text{Q}{}_{\mathrm{B}}\text{→}\text{hv}\text{P}{}^{\mathrm{+}}\text{Q}{}_{\mathrm{A}}\text{Q}{}^{\mathrm{?}}{}_{\mathrm{B}}$, following flash excitation of photosynthetic reaction centers isolated from Rhodopseudomonas sphaeroides. P is a bacteriochlorophyll dimer (P-870), and Q_A and Q_B are ubiquinones. In reaction centers containing only Q_A, the enthalpy of P⁺Q^?_A is very close to that of the PQ_A ground state (ΔH_r = 0.05 ± 0.03 eV). The free energy of about 0.65 eV that is captured in the photochemical reaction evidently takes the form of a substantial entropy decrease. In contrast, the formation of P⁺Q_AQ^?_B in reaction centers containing both quinones has a ΔH_r of 0.32 ± 0.02 eV. The entropy change must be near zero in this case. In the presence of o-phenanthroline, which blocks electron transfer between Q^?_A and Q_B, ΔH_r for forming P⁺Q^?_AQ_B is 0.13 ± 0.03 eV. The influence of flash-induced proton uptake on the results was investigated, and the ΔH_r values given above were measured under conditions that minimized this influence. Although the reductions of Q_A and Q_B involve very different changes in enthalpy and entropy, both reactions are accompanied by a similar volume decrease of about 20 ml/mol. The contraction probably reflects electrostriction caused by the charges on P⁺ and Q^?_A or Q^?_B.     

Equilibrium constants under physiological conditions for the reactions of L-phosphoserine phosphatase and pyrophosphate: L-serine phosphotransferase

The observed equilibrium constants (K_obs) for the l-phosphoserine phosphatase reaction [EC 3.1.3.3] have been determined under physiological conditions of temperature (38 °C) and ionic strength (0.25 m) and physiological ranges of pH and free [Mg²⁺]. Using Σ and square brackets to indicate total concentrations $\text{K}{}_{\mathrm{<mtext>obs</mtext>}}\text{=}\text{Σ L-serine][Σ P}{}_{\mathrm{i}}\text{]}\text{Σ L-phosphoserine]H}{}_2\text{O]}\text{, K =}\text{L-H · serine}{}^{\mathrm{\pm }}\text{]HPO}{}_4{}^{\mathrm{2?}}\text{]}\text{[L-H · phosphoserine}{}^{\mathrm{2?}}\text{]H}{}_2\text{O]}$. The value of K_obs has been found to be relatively sensitive to pH. At 38 °C, K⁺] = 0.2 m and free [Mg²⁺] = 0; K_obs = 80.6 m at pH 6.5, 52.7 m at pH 7.0 [ΔG_obs⁰ = ?10.2 kJ/mol (?2.45 kcal/mol)], and 44.0 m at pH 8.0 ([H₂O] = 1). The effect of the free [Mg²⁺] on K_obs was relatively slight; at pH 7.0 ([K⁺] = 0.2 m) K_obs = 52.0 m at free [Mg²⁺] = 10^?3, m and 47.8 m at free [Mg²⁺] = 10^?2, m. K_obs was insignificantly affected by variations in ionic strength (0.12–1.0 m) or temperature (4–43 °C) at pH 7.0. The value of K at 38 °C and I = 0.25 m has been calculated to be 34.2 ± 0.5 m [ΔG_obs⁰ = ?9.12 kJ/mol (?2.18 kcal/ mol)]([H₂O] = 1). The K for the phosphoserine phosphatase reaction has been combined with the K for the reaction of inorganic pyrophosphatase [EC 3.6.1.1] previously estimated under the same physiological conditions to calculate a value of 2.04 × 10⁴, m [ΔG_obs⁰ = ?28.0 kJ/mol (?6.69 kcal/mol)] for the K of the pyrophosphate:l-serine phosphotransferase [EC 2.7.1.80] reaction. $\text{K}{}_{\mathrm{<mtext>obs</mtext>}}\text{=}\text{[}\text{Σ L-serine}\text{][}\text{Σ}\text{P}{}_{\mathrm{<mtext>i</mtext>}}\text{]}\text{[}\text{Σ L-phosphoserine}\text{][}\text{H}{}_2\text{O}\text{]}\text{, K =}\text{[L-H · serine}{}^{\mathrm{\pm }}\text{]HPO}{}_4{}^{\mathrm{2?}}\text{]}\text{[L-H · phosphoserine}{}^{\mathrm{2?}}\text{]H}{}_2\text{O}$. Values of K_obs for this reaction at 38 °C, pH 7.0, and I = 0.25 m are very sensitive to the free [Mg²⁺], being calculated to be 668 [ΔG_obs⁰ = ?16.8 kJ/mol (?4.02 kcal/mol)] at free [Mg²⁺] = 0; 111 [ΔG_obs⁰ = ?12.2 kJ/mol (?2.91 kcal/mol)] at free [Mg²⁺] = 10^?3, m; and 9.1 [ΔG_obs⁰ = ?5.7 kJ/mol (?1.4 kcal/mol) at free [Mg²⁺] = 10^?2, m). K_obs for this reaction is also sensitive to pH. At pH 8.0 the corresponding values of K_obs are 4000 [ΔG_obs⁰ = ?21.4 kJ/mol (?5.12 kcal/mol)] at free [Mg²⁺] = 0; and 97.4 [ΔG_obs⁰ = ?11.8 kJ/ mol (?2.83 kcal/mol)] at free [Mg²⁺] = 10^?3, m. Combining K_obs for the l-phosphoserine phosphatase reaction with K_obs for the reactions of d-3-phosphoglycerate dehydrogenase [EC 1.1.1.95] and l-phosphoserine aminotransferase [EC 2.6.1.52] previously determined under the same physiological conditions has allowed the calculation of K_obs for the overall biosynthesis of l-serine from d-3-phosphoglycerate. $\text{K}{}_{\mathrm{<mtext>obs</mtext>}}\text{=}\text{[}\text{Σ L-serine}\text{][}\text{Σ NADH}\text{][}\text{Σ}\text{P}{}_{\mathrm{<mtext>i</mtext>}}\text{][}\text{Σ}\text{α-}\text{ketoglutarate}\text{]}\text{[Σ d-3-}\text{phosphoglycerate}\text{][Σ NAD}{}^{\mathrm{+}}\text{][Σ L-glutamat0]}$ The value of K_obs for these combined reactions at 38 °C, pH 7.0, and I = 0.25 m (K⁺ as the monovalent cation) is 1.34 × 10^?2, m at free [Mg²⁺] = 0 and 1.27 × 10^?2, m at free [Mg²⁺] = 10^?3, m.     

Kinetic studies on the reaction of ethylenediaminetetraacetatochromium(III) complex with hydrogen peroxide

The rate of reaction of [Cr(III)Y]_aq (Y is EDTA anion) with hydrogen peroxide was studied in aqueous nitrate media [μ = 0.10 M (KNO₃)] at various temperatures. The general rate equation, Rate = $\text{k}{}_1\text{+}\text{k}{}_2\text{K}{}_1\text{[H}{}^{\mathrm{+}}\text{]}{}^{\mathrm{?1}}\text{1 +}\text{K}{}_1\text{[H}{}^{\mathrm{+}}\text{]}{}^{\mathrm{?1}}$ [Cr(III)Y]_aq[H₂O₂] holds over the pH range 5–9. The decomposition reaction of H₂O₂ is believed to proceed via two pathways where both the aquo and hydroxo-quinquedentate EDTA complexes are acting as the catalyst centres. Substitution-controlled mechanisms are suggested and the values of the second-order rate constants k₁ and k₂ were found to be 1.75 × 10^?2 M^?1 s^?1 and 0.174 M^?1 s^?1 at 303 K respectively, where k₂ is the rate constant for the aquo species and k₂ is that for the hydroxo complex. The respective activation enthalpies (ΔH^*₁ = 58.9 and ΔH^*₂ = 66.5 KJ mol^?1) and activation entropies (ΔS^*₁ = ?85 and ΔS^*₂ = ?40 J mol^?1 deg^?1) were calculated from a least-squares fit to the Eyring plot. The ionisation constant pK₁, was inferred from the kinetic data at 303 K to be 7.22. Beyond pH 9, the reaction is markedly retarded and ceases completely at pH ? 11. This inhibition was attributed in part to the continuous loss of the catalyst as a result of the simultaneous oxidation of Cr(III) to Cr(VI).     

Energy-coupling mechanisms under aerobic and anaerobic conditions in autotrophically grown Pseudomonas saccharophila

The cell-free preparations from autotrophieally grown Pseudomonas saccharophila catalyzed the process of electron transport from H₂ or various other organic electron donors to either O₂ or NO₃^? with concomitant ATP generation. The respective $\text{P}\text{O}$ ratios with H₂ and NADH were 0.63 and 0.73, the respective $\text{P}\text{NO}{}_3{}^{\mathrm{?}}$ ratios were 0.57 and 0.54. In contrast, the $\text{P}\text{O}$ and $\text{P}\text{NO}{}_3{}^{\mathrm{?}}$ ratios with succinate were 0.18 and 0.11, respectively. ATP formation coupled to the oxidation of ascorbate, in the absence or presence of added N,N,N′,N′-tetramethyl-p-phenylenediamine or cytochrome c, could not be detected. Various uncouplers inhibited phosphorylation with either O₂ or NO₃^? as terminal electron acceptors without affecting the oxidation of H₂ or other substrates. The NADH oxidation at the expense of O₂ or NO₃^? reduction as well as the associated phosphorylation were inhibited by rotenone and amytal. The aerobic and anaerobic H₂ oxidation and coupled ATP synthesis, on the other hand, was unaffected by the flavoprotein inhibitors as well as by the NADH trapping system. The NADH, H₂, and succinate-linked electron transport to O₂ or NO₃^? and the associated phosphorylations were sensitive, however, to antimycin A or 2-n-nonyl-4-hydroxyquino-line-N-oxide, and cyanide or azide. The data indicated that although the phosphorylation sites 1 and II were associated with NADH oxidation by O₂ or NO₃^?, the energy conservation coupled to H₂ oxidation under aerobic or anaerobic conditions appeared to involve site II only.     

Respiration-driven proton translocation with nitrite and nitrous oxide in Paracoccus denitrificans

(1) $\text{H}$⁺/electron acceptor ratios have been determined with the oxidant pulse method for cells of denitrifying Paracoccus denitrificans oxidizing endogenous substrates during reduction of O₂, NO^?₂ or N₂O. Under optimal H⁺-translocation conditions, the ratios $\text{H}{}^{\mathrm{+}}\text{O}$, $\text{H}{}^{\mathrm{+}}\text{N}{}_2\text{O}$, $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂ and $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂O were 6.0–6.3, 4.02, 5.79 and 3.37, respectively. (2) With ascorbate/N,N,N′,N′-tetramethyl-p-phenylenediamine as exogenous substrate, addition of NO^?₂ or N₂O to an anaerobic cell suspension resulted in rapid alkalinization of the outer bulk medium. $\text{H}{}^{\mathrm{+}}\text{N}{}_2\text{O}$, $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂ and $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂O were ?0.84, ?2.33 and ?1.90, respectively. (3) The $\text{H}{}^{\mathrm{+}}\text{oxidant}$ ratios, mentioned in item 2, were not altered in the presence of $\text{valinomycin}\text{K}{}^{\mathrm{+}}$ and the triphenylmethylphosphonium cation. (4) A simplified scheme of electron transport to O₂, NO^?₂ and N₂O is presented which shows a periplasmic orientation of the nitrite reductase as well as the nitrous oxide reductase. Electrons destined for NO^?₂, N₂O or O₂ pass two H⁺-translocating sites. The $\text{H}{}^{\mathrm{+}}\text{electron}$ acceptor ratios predicted by this scheme are in good agreement with the experimental values.     

Characterization of sodium and proton flows in sub-bacterial particles of Halobacterium halobium in terms of nonequilibrium thermodynamics

A thermodynamic characterization of the Na⁺-H⁺ exchange system in Halobacterium halobium was carried out by evaluating the relevant phenomenological parameters derived from potential-jump measurements. The experiments were performed with sub-bacterial particles devoid of the purple membrane, in 1 M NaCl, 2 M KCl, and at pH 6.5–7.0. Jumps in either pH or pNa were brought about in the external medium, at zero electric potential difference across the membrane, and the resulting relaxation kinetics of protons and sodium flows were measured. It was found that the relaxation kinetics of the proton flow caused by a pH-jump follow a single exponential decay, and that the relaxation kinetics of both the proton and the sodium flows caused by a pNa-jump also follow single exponential decay patterns. In addition, it was found that the decay constants for the proton flow caused by a pH-jump and a pNa-jump have the same numerical value. The physical meaning of the decay constants has been elucidated in terms of the phenomenological coefficients (mobilities) and the buffering capacities of the system. The phenomenological coefficients for the Na⁺-H⁺ flows were determined as differential quantities. The value obtained for the total proton permeability through the particle membrane via all available channels, $\text{L}{}_{\mathrm{<mtext>H</mtext>}}\text{= (}\text{?J}{}_{\mathrm{<mtext>H\; +</mtext>}}\text{?Δ}\text{pH}\text{)}{}_{\mathrm{<mtext>\Delta \psi ,\Delta </mtext><mtext>pNa</mtext>}}$, was in the range of 850–1150 nmol H⁺·(mg protein)^?1·h^?1·(pH unit)^?1 for four different preparations; for the total Na⁺ permeability, $\text{L}{}_{\mathrm{<mtext>Na</mtext>}}\text{= (}\text{?J}{}_{\mathrm{<mtext>Na</mtext>}}{}^{\mathrm{+}}\text{?Δ}\text{pNa}\text{)}{}_{\mathrm{<mtext>\Delta \psi ,\Delta </mtext><mtext>pH</mtext>}}$, it was 1620–2500 nmol Na⁺·(mg protein)^?1·h^?1·(pNa unit)^?1; and for the proton ‘cross-permeability’, $\text{L}{}_{\mathrm{<mtext>HNa</mtext>}}\text{= (}\text{?J}{}_{\mathrm{<mtext>H</mtext>}}{}^{\mathrm{+}}\text{?Δ}\text{pNa}\text{)}{}_{\mathrm{<mtext>\Delta \psi ,\Delta </mtext><mtext>pH</mtext>}}$, it was 220–580 nmol H⁺·(mg protein)^?1·h^?1·(pNa unit)^?1, for different preparations. From the above phenomenological parameters, the following quantities have been calculated: the degree of coupling (q), the maximal efficiency of Na⁺-H⁺ exchange (η_max), the flow and force efficacies (?) of the above exchange, and the admissible range for the values of the molecular stoichiometry parameter (r). We found q ? 0.4; η_max ? 5%; 0.36 ? r ? 2; ?_JNa⁺ ? 1.3 · 10⁵μmol · (RT unit)^?1 at J_Na = 1 μmolNa⁺ · (mgprotein)^?1 · h^?1; and ?_ΔpNa ? 5 · 10⁴ ΔpNa · (mg protein) · h · (RT unit)^?1 at ΔpNa = 1 unit, for different preparations.     

Nucleotide exchange and control of ATPase activity in Rhodospirillum rubrum chromatophores

(1) Light-activated ‘dark’ ATPase in Rhodospirillum rubrum chromatophores is inhibited by preincubation with ADP or ATP (in the absence of Mg²⁺). I₅₀ values were 0.5 and 6 μM, respectively, after 20 s of preincubation. (2) In the absence of MgATP, the rate constant for dissociation of ADP or ATP from the inhibitory site was less than 0.2 min^?1 in deenergized membranes. Illumination in the absence of MgATP caused an increase of over 60-fold in both rate constants. (3) In some experiments hydrolysis was performed in the presence of 10 μM Mg²⁺ and 0.2 mM MgATP. Under these conditions, the ADP or ATP inhibition was reversed within about 20 or about 80 s, respectively, after the onset of hydrolysis. This suggests that recovery from ADP or ATP inhibition (i.e., release of tightly bound ADP or ATP) in the dark is induced by MgATP binding to a second nucleotide-binding site on the enzyme. (4) Results obtained with variable concentrations of uncoupler suggest that in the absence of bound Mg²⁺ (see below), MgATP-induced release of tightly bound ADP or ATP does not require a transmembrane . This, together with the inhibitor/substrate ratios prevalent during hydrolysis, suggests that these reactivation reactions involve MgATP binding to a high-affinity binding site (Kd < 2 μM). (5) At high concentrations of uncoupler, a time-dependent inhibition of hydrolysis occurred in the control chromatophores as well as in the nucleotide-pretreated chromatophores. This deactivation was dependent on Mg²⁺. In addition, MgATP-dependent reversal of ADP inhibition in the dark was inhibited by Mg²⁺ at concentrations above 20–30 μM. By contrast, MgATP-dependent reversal of ADP inhibition occurs within 3–4 s, despite the presence of high concentrations of Mg²⁺ if the chromatophores are illuminated during contact with the nucleotides. Uncoupler abolishes the effect of illumination. A reaction scheme incorporating these findings is proposed. (6) The implications of these findings for the mechanism of lightactivation of ATP hydrolysis (Slooten, L. and Nuyten, A., (1981) Biochim. Biophys. Acta 638, 305–312) are discussed.     

Complete stoichiometry of free NADPH oxidation in liver microsomes

The stoichiometry of free NADPH oxidation in phenobarbital induced rabbit liver microsomes was measured by means of registering the rates of NADPH, H⁺ and O₂ consumption and $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ and H₂O₂ production. $\text{ΔO}{}_2{}^{\mathrm{<mtext>?</mtext>}}\text{:ΔH}{}_2\text{O}{}_2$ ratio is approximately I indicating that about half H₂O₂ results from $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ dismutation, the second half being formed directly. ΔNADPH:ΔH₂O₂ and ΔO₂:ΔH₂O₂ ratios exceed I and therefore another product of the reaction is water. The fact that the ratio (ΔNADPH-ΔH₂O₂):(ΔO₂-ΔH₂O₂) is 2 allows one to consider direct 4-electron O₂ reduction as the major way of water formation rather than endogenous substrate hydroxylation.     

A thermodynamic description of the self-association of flavin mononucleotide.

Systematic heat of dilution studies of the self-association of flavin mononucleotide (FMN) have been conducted as a function of ionic strength (0.05 – 2.0 m) and pH (5–9) in aqueous solution. The data are adequately described by the expression $\text{Q}{}_{\mathrm{T}}\text{= ΔH ? (}\text{ΔH}\text{K}\text{)}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{(}\text{Q}{}_{\mathrm{T}}\text{c}{}_{\mathrm{T}}\text{)}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for an isodesmic self-association. Q_T is the molar heat of dilution, ΔH and K are the derived enthalpy and equilibrium constants for the process FMN + (FMN)_i?1 ? (FMN)_i, and c_T is the concentration of FMN expressed in monomer units. Typical values derived for the various thermodynamic parameters at 25 °C are ΔG = ?3.56 kcal mol^?1, ΔH = ?3.72 kcal mol^?1, and ΔS = ?0.54 cal (mol · deg)^?1. These data, plus nuclear magnetic resonance evidence (Yagi, K., Ohishi, N., Takai, A., Kawano, K., and Kyogoku, Y., 1976, Biochemistry15, 2877–2880) argue in favor of an open-ended association of flavin molecules. The signs of the various thermodynamic parameters suggest that both hydrophobic and surface energy forces contribute significantly to the association, while the lack of any significant ionic strength dependence indicates the lack of any ionic centers in the association.     

Differences in uncoupling effects associated with the uptake of lactose and dansyl-galactoside in Escherichia coli membrane: Active transport versus specific binding

Cytoplasmic membrane vesicles isolated from Escherichia coli take up dansyl-galactoside, a fluorescent competitive inhibitor of lactose transport, to much lower levels than lactose. An initial interpretation, based on the study of the fluorescent changes accompanying the energy-dependent uptake, was that it represented a one-to-one specific binding to the lac carrier protein which was not followed by transport. Recently, on the basis of a new estimation of the number of lac carrier in the membrane, it has been advanced that the uptake of dansyl-galactoside represents a nonspecific binding on the inner surface of the membrane following transport. We discriminate between the two interpretations by comparing the effects of lactose and dansyl-galactoside uptake on the electrochemical gradient of protons ($\text{Δ}\text{}\text{?}\text{gm}{}_{\mathrm{H}}\text{+}$), generated by the oxidation of substrates, and on the uptake of proline. Indeed, it is known that the rate of lactose transport is such that it leads, as a consequence of the lactose/H⁺ symport, to an observable decrease of $\text{Δ}\text{}\text{?}\text{gm}{}_{\mathrm{H}}\text{+}$, and secondary to this decrease to an inhibition of the uptake of proline transported at much lower rate. We show that the rates of uptake of lactose and dansyl-galactoside by the membrane vesicles are similar; yet the uptake of dansyl-galactoside does not lead to the uncoupling effects which are associated with the uptake of lactose. We discuss the possible reasons for the absence of this uncoupling effect, and we conclude that our data are incompatible with the notion that the energy-dependent uptake of dansyl-galactoside is associated with an active transport involving a dansyl-galactoside/H⁺ symport. On the contrary, the data substantiate the initial interpretation that the energy-dependent uptake of dansyl-galactoside reflects the binding to the lac carrier not followed by transport.     

An estimation of the light-induced electrochemical potential difference of protons across the membrane of Halobacterium halobium

The light-dependent uptake of triphenylmethylphosphonium (TPMP⁺) and of 5,5-dimethyloxazolidine-2,4-dione (DMO) by starved purple cells of Halobacterium halobium was investigated. DMO uptake was used to calculate the pH difference (ΔpH) across the membrane, and TPMP⁺ was used as an index of the electrical potential difference, Δψ.Under most conditions, both in the light and in the dark, the cells are more alkaline than the medium. In the light at pH 6.6, ΔpH amounts to 0.6–0.8 pH unit. Its value can be increased to 1.5–2.0 by either incubating the cells with TPMP⁺ (10^?3 M) or at low external pH (5.5). — ΔpH can be lowered by uncoupler or by nigericin. The TPMP⁺ uptake by the cells indicates a large Δψ across the membrane, negative inside. It was estimated that in the light, at pH 6.6, Δψ might reach a value of about 100 mV and that consequently the electrical equivalent of the proton electrochemical potential difference, , amounts under these conditions to about 140 mV.The effects of different ionophores on the light-driven proton extrusion by the cells were in agreement with the effects of these compounds on — ΔpH.     

Thiophosphorylation of (Na + K+)-ATPase yields an ADP-sensitive phosphointermediate

(1) Treatment of $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ from rabbit kidney outer medulla with the $\text{γ-}{}^{35}\text{S}$ labeled thio-analogue of ATP in the presence of $\text{Na}{}^{\mathrm{+}}\text{+ Mg}{}^{\mathrm{2+}}$ and the absence of K⁺ leads to thiophosphorylation of the enzyme. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for [γ-S]ATP is 2.2 μM and for Na⁺ 4.2 mM at 22°C. Thiophosphorylation is a sigmoidal function of the Na⁺ concentration, yielding a Hill coefficient $\text{n}\text{H}\text{= 2.6}$. (2) The thio-analogue ($\text{K}{}_{\mathrm{<mtext>m</mtext><mtext>\; =\; 35\; \mu M</mtext>}}$) can also support overall $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ activity, but $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ at 37°C is only $\text{1.3 γ}\text{mol}\text{· (mg protein)}{}^{\mathrm{?}}\text{·}$ h^?1 or 0.09% of the specific activity for ATP ($\text{K}{}_{\mathrm{<mtext>m</mtext><mtext>\; =\; 0.43\; mM</mtext>}}$). (3) The thiophosphoenzyme intermediate, like the natural phosphoenzyme, is sensitive to hydroxylamine, indicating that it also is an acylphosphate. However, the thiophosphoenzyme, unlike the phosphoenzyme, is acid labile at temperatures as low as 0°C. The acid-denatured thiophosphoenzyme has optimal stability at pH 5–6. (4) The thiophosphorylation capacity of the enzyme is equal to its phosphorylation capacity, indicating the same number of sites. Phosphorylation by ATP excludes thiophosphorylation, suggesting that the two substrates compete for the same phosphorylation site. (5) The (apparent) rate constants of thiophosphorylation (0.4 s^?1 vs. 180 s^?1), spontaneous dethiophosphorylation (0.04 s^?1 vs. 0.5 s^?1) and K⁺-stimulated dethiophosphorylation (0.54 s^?1 vs. 230 s^?1) are much lower than those for the corresponding reactions based on ATP. (6) In contrast to the phosphoenzyme, the thiophosphoenzyme is ADP-sensitive (with an apparent rate constant in ADP-induced dethiophosphorylation of 0.35 s^?1, $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$$\text{ADP = 48 μM at 0.1 mM ATP}$) and is relatively K⁺-insensitve. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for K⁺ in dethiophosphorylation is 0.9 mM and in dephosphorylation 0.09 mM. The thiophosphoenzyme appears to be for 75–90% in the ADP-sensitive E₁-conformation.     

Ultracentrifugation studies on early stage polymerization of tobacco mosaic virus protein

The effects of absolute temperature (T), ionic strength (μ), and pH on the polymerization of tobacco mosaic virus protein from the 4 S form (A) to the 20 S form (D) were investigated by the method of sedimentation velocity. The loading concentration in grams per liter (C) was determined at which a just-detectable concentration (β) of 20 S material appeared. It was demonstrated experimentally that under the conditions employed herein, an equilibrium concentration of 20 S material was achieved in 3 h at the temperature of the experiment and that 20 S material dissociated again in 4 h or less to 4 S material either upon lowering the temperature or upon dilution. Thus, the use of thermodynamic equations for equilibrium processes was shown to be valid. The equation used to interpret the results, log ($\text{C?β) =}\text{constant}\text{+ (}\text{ΔH}{}^1\text{2.3RT}$) + ($\text{Δ}\text{W}{}^1{}_{\mathrm{<mtext>el</mtext>}}\text{2.3RT}$) ? K′_sμ + ζpH, was derived from three separate models of the process, the only difference being in the anatomy of the constant; thus, the method of analysis is essentially independent of the model. $\text{ΔH}{}^1$ and ΔW¹_el are the enthalpy and the change in electrical work per mole of A protein (the trimer of the polypeptide chain), K^′_s is the salting-out constant on the ionic strength basis, ζ is the number of moles of hydrogen ion bound per mole of A protein in the polymerization, and R is the gas constant. The three models leading to this equation are: a simple 11th-order equilibrium between A₁ (the trimer of the polypeptide chain) and D, either the double disk or the double spiral of approximately the same molecular weight, designated model A; a second model, designated B, in which A₁ was assumed to be in equilibrium with D at the same time that it is in equilibrium with A₂, A₃, etc., dimers and trimers, etc., of A₁ in an isodesmic system; and a phase-separation model, designated model C, in which A protein is treated as a soluble material in equilibrium with D, considered as an insoluble phase. From electrical work theory, $\text{Δ}\text{W}{}_{\mathrm{el}}{}^1\text{/T}$ was shown to be essentially independent of T; therefore, in experiments at constant μ and constant pH the equation of log (C ? β) versus 1/T is linear with a slope of $\text{ΔH}{}^1\text{/2.3R}$. The results fit such an equation over nearly a 20 °C-temperature range with a single value of $\text{Δ}\text{H}{}^1$ of +32 kcal/mol A₁. Results obtained when T and pH were held constant but μ was varied did not fit a straight line, which shows that more than simple salting-out is involved. When the effect of ionic strength on the electrical work contribution was considered in addition to salting-out, the data were interpreted to indicate a value of $\text{Δ}\text{W}{}^1{}_{\mathrm{el}}$ of 1.22 kcal/mol A₁ at pH 6.7 and a value of 4.93 for K_s^′. When μ and T were held constant but pH was varied, and when allowance was made for the effect of pH changes on the electrical work contribution, a value of 1.1 was found for ζ. This means that something like 1.1 mol of hydrogen ion must be bound per mole of A₁ protein in the formation of D. When this is added to the small amount of hydrogen ion bound per A₁ before polymerization, at the pH values used, it turned out that for D to be formed, 1.5 H⁺ ions must be bound per A₁ or 0.5 per protein polypeptide chain. This amounts to 1 H⁺ ion per polypeptide chain for half of the protein units, presumably those in one but not the other layer of the double disk or turn of the double spiral. When polymerization goes beyond the D stage, as shown by previously published data, additional H⁺ ions are bound. Simultaneous osmotic pressure studies and sedimentation studies were carried out, in both cases as a function of loading concentration C. These results were in complete disagreement with models A and C but agreed reasonably well with model B. The sedimentation studies permitted evaluation of the constant, β, to be 0.33 g/liter.     

Photosystem II energy coupling in chloroplasts with H2O2 as electron donor

NH₂OH-treated, non-water-splitting chloroplasts can oxidize H₂O₂ to O₂ through Photosystem II at substantial rates (100–250 μequiv · h^?1 · mg^?1 chlorophyll with 5 mM H₂O₂) using 2,5-dimethyl-p-benzoquinone as an electron acceptor in the presence of the plastoquinone antagonist dibromothymoquinone. This H₂O₂ → Photosystem II → dimethylquinone reaction supports phosphorylation with a $\text{P}\text{e}{}_2$ ratio of 0.25–0.35 and proton uptake with $\text{H}{}^{\mathrm{+}}\text{e}$ values of 0.67 (pH 8)–0.85 (pH 6). These are close to the $\text{P}\text{e}{}_2$ value of 0.3–0.38 and the $\text{H}{}^{\mathrm{+}}\text{e}$ values of 0.7–0.93 found in parallel experiments for the H₂O → Photosystem II → dimethylquinone reaction in untreated chloroplasts. Semi-quantitative data are also presented which show that the donor → Photosystem II → dibromothymoquinone (→O₂) reaction can support phosphorylation when the donor used is a proton-releasing reductant (benzidine, catechol) but not when it is a non-proton carrier (I^?, ferrocyanide).     

The accumulation of superoxide radical during the aerobic action of xanthine oxidase A requiem for H2O4−

The action of xanthine oxidase upon acetaldehyde or xanthine at pH 10.2 has been shown to be accompanied by substantial accumulation of O₂^? during the first few minutes of the reaction. H₂O₂ decreases this accumulation of O₂^? presumably because of the Haber-Weiss reaction ($\text{H}{}_2\text{O}{}_2\text{+}\text{O}{}_2{}^{\mathrm{?}}\text{→}\text{OH}{}^{\mathrm{?}}\text{+}\text{OH}\text{+}\text{O}{}_2$) and very small amounts of superoxide dismutase eliminate it. This accumulation of O₂^? was demonstrated in terms of a burst of reduction of cytochrome $\text{c}$, seen when the latter compound was added after aerobic preincubation of xanthine oxidase with its substrate. The kinetic peculiarities of the luminescence seen in the presence of luminol, which previously led to the proposal of H₂O₄^?, can now be satisfactorily explained entirely on the basis of known radical intermediates.