Norman E. Good

Hydrogen ion buffers for biological research

Five new zwitterionic hydrogen ion buffers are described for the first time. These buffers, all N-substituted 3-amino-2-hydroxypropanesulfonic acids, with pKas between 6.9 and 7.9 have been subjected to a number of stringent tests in mammalian tissue culture, plant pathology, and virology to detect inhibitory effects. They appear to be equivalent to or better than any other buffers heretofore available.

The stoichiometry of photophosphorylation

Abstract Using chloroplasts from three different plant species in the presence of four different buffer systems we found that the P 2e ratio was consistently above 1.0 if the pH was between 8.4 and 9.4. At pH 8.9 the ratio was usually above 1.25 and indeed no single determination of the ratio fell below 1.1 at this pH unless Tris-HCl was present. Even if the endogenous phosphorylation were subtracted (a procedure which is probably not valid) no single determination would yield a value of P 2e as low as 1.0. Consequently it seems very probable that the theoretical maximum efficiency of photophosphorylation is higher than has been thought.

Photophosphorylation as a function of illumination time. II. Effects of permeant buffers

(1) The amounts of orthophosphate, bicarbonate and tris (hydroxymethyl)-aminomethane found inside the thylakoid are almost exactly the amounts predicted by assuming that the buffers equilibrate across the membrane. Since imidazole and pyridine delay the development of post-illumination ATP formation while increasing the maximum amount of ATP formed, it follows that such relatively permeant buffers must also enter the inner aqueous space of the thylakoid. (2) Photophosphorylation begins abruptly at full steady-state efficiency and full steady-state rate as soon as the illumination time exceeds about 5 ms when permeant ions are absent or as soon as the time exceeds about 50 ms if valinomycin and KC1 are present. In either case, permeant buffers have little or no effect on the time of illumination required to initiate phosphorylation. A concentration of bicarbonate which would delay acidification of the bulk of the inner aqueous phase for at least 350 ms has no effect at all on the time of initiation of phosphorylation. In somewhat swollen chloroplasts, the combined buffering by the tris(hydroxymethyl) aminomethane and orthophosphate inside would delay acidification of the inside by 1500 ms but, even in the presence of valinomycin and KC1, the total delay in the initiation of phosphorylation is then only 65 ms. Similar discrepancies occur with all of the other buffers mentioned. (3) Since these discrepancies between internal acidification and phosphorylation are found in the presence of saturating amounts of valinomycin and KC1, it seems that photophosphorylation can occur when there are no proton concentration gradients and no electrical potential differences across the membranes which separate the medium from the greater part of the internal aqueous phase. (4) We suggest that the protons produced by electron transport may be used directly for phosphorylation without even entering the bulk of the inner aqueous phase of the lamellar system. If so, phosphorylation could proceed long before the internal pH reflected the proton activity gradients within the membrane.

The number of sites sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea,3-(4-chlorophenyl)-1,1-dimethylurea and 2-chloro-4-(2-propylamino)-6-ethylamino-s-triazine in isolated chloroplasts.

Abstract The absorption of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, 3-(4-chlorophenyl)-1,1-dimethylurea and Atrazine by isolated spinach chloroplasts involves at least three simultaneous processes: (a) An irreversible binding or destruction accounting for about one molecule of inhibitor for every 1000 chlorophyll molecules. This process is not associated with inhibition. (b) A partitioning between the biological and aqueous solvent phases which is independent of inhibitor concentration over the inhibitory range. (c) An absorption which corresponds closely to the degree of inhibition. This process probably represents the formation of the enzyme-inhibitor complex. Estimates of the number of inhibitor-sensitive sites based on the analysis of the partitioning phenomena and estimates based on the Straus-Goldstein analysis of the inhibition kinetics agree. Regardless of which of the three inhibitors is used the number of sites of inhibition seems to be one for every 2500 chlorophyll molecules. The nature of the “photosynthetic unit” is discussed and the question of the minimum size of chloroplast fragments capable of oxygen production is reconsidered in the light of these observations.

Electron transport and photophosphorylation in chloroplasts as a function of the electron acceptor. III. A dibromothymoquinone-insensitive phosphorylation reaction associated with Photosystem II

Abstract Dibromothymoquinone (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone) is reputed to be a plastoquinone antagonist which prevents the photoreduction of hydrophilic oxidants such as ferredoxin-NADP+. However, we have found that dibromothymoquinone inhibits only a small part of the photoreduction of lipophilic oxidants such as oxidized p-phenylenediamine. Dibromothymoquinone-resistant photoreduction reactions are coupled to phosphorylation, about 0.4 molecules of ATP consistently being formed for every pair of electrons transported. Dibromothymoquinone itself is a lipophilic oxidant which can be photoreduced by chloroplasts, then reoxidized by ferricyanide or oxygen. The electron transport thus catalysed also supports phosphorylation and the P e 2 ratio is again 0.4. It is concluded that there is a site of phosphorylation before the dibromothymoquinone block and another site of phosphorylation after the block. The former site must be associated with electron transfer reactions near Photosystem II, while the latter site is presumably associated with the transfer of electrons from plastoquinone to cytochrome f.

The stoichiometric relation of phosphorylation to electron transport in isolated chloroplasts

Abstract Electron transport in chloroplasts can proceed by two pathways. One pathway is dependent on the presence of ADP and orthophosphate and is responsible for photophosphorylation. This pathway is sensitive to the photophosphorylation inhibitor phlorizin, to antimycin A and to p -chloromercuribenzoate (PCMB). The other pathway is independent of phosphorylation, insensitive to phlorizin, relatively insensitive to antimycin A and insensitive to PCMB. Apparently amines increase the electron transport rate by uncoupling the antimycin-sensitive phosphorylating pathway while carbonylcyanide phenylhydrazones uncouple by increasing the activity of the antimycin-resistant non-phosphorylating pathway. During photophosphorylation the electron transport rate normally exceeds the corresponding rate measured in the absence of phosphate and, under many conditions, the amount of ATP formed is proportional to the additional electron transport which occurs in the presence of phosphate. Indeed there is often a rather precise mole for mole correspondence between ATP formation and the accompanying increase in ferricyanide reduction. This is true when phosphorylation conditions are optimal and it is also true when the phosphorylation rate is varied over a wide range by limiting concentrations of phosphate, by the addition of antimycin A, by the addition of PCMB, or by the addition of carbonylcyanide phenylhydrazones. It is not true unless the rate of electron transport is controlled at the level of the phosphorylation reaction. Thus the correspondence between phosphorylation and extra electron transport is no longer observed when electron transport is limited by low light intensities or by the addition of phenylureas. Under these conditions phosphorylation may greatly exceed the increase in electron transport associated with phosphorylation. Moreover the correspondence is not always found when the chloroplasts have been partially uncoupled by amines such as Tris or methylamine. Our observations suggest that, at high light intensities and in the absence of amine uncouplers, the phosphorylating electron transport is superimposed on a constant non-phosphorylating electron transport. If this is so the phosphorylating process must yield two molecules of ATP for every pair of electron it transfers.

Energy thresholds for ATP synthesis in chloroplasts

Abstract We have investigated the ATP synthesis associated with acid-base transitions in chloroplast lamellae under conditions which allow simultaneous control of the thermodynamic variables, ΔpH, membrane potential and ΔGATP. These variables have been directly imposed rather than simply inferred. Since the initiation of labeled Pi incorporation seems to measure accurately the initiation of net ATP synthesis, the following conclusions can be drawn: (1) The proton-motive force which is just sufficient for ATP synthesis provides almost exactly the required energy for ΔGATP if the efflux of three H+ is required for each ATP molecule formed. (2) The membrane potential and the ΔpH contribute to the proton-motive force in a precisely additive way. Thus, the threshold can be reached or exceeded by a ΔpH in the absence of a membrane potential, by a membrane potential in the absence of a ΔpH, or by any combination of membrane potential and ΔpH. With a large enough membrane potential, ATP synthesis occurs even against a small inverse ΔpH. In each instance the combined ΔpH and membrane potential necessary for initiation of ATP synthesis represent the same threshold proton-motive force.

Uncoupling and Energy Transfer Inhibition in Photophosphorylation

Publisher Summary This chapter discusses uncoupling and energy transfer inhibition in photophosphorylation. The uncouplers promote non-phosphorylating electron transport and this lowers the efficiency of phosphorylation. During light-induced electron transport at low pH, chloroplasts shrink, make the medium more alkaline, and acquire the capacity to form adenosine triphosphate (ATP) in a subsequent dark period. Uncouplers might modify the conformational changes, cause rapid hydrogen ion equilibration across the chloroplast membranes, and speed the decay of the ATP-synthesizing ability. Electron transport—which takes place under the conditions that are optimal for phosphorylation—results in conformational change. The same is true of electron transport uncoupled by ethylenediaminetetraacetic acid (EDTA) treatment. The carbonyl cyanide phenylhydrazone uncouplers abolish all conformational changes. Amine-uncoupled electron transport results in chloroplast swelling while atebrin- and chlorpromazine-uncoupled electron transport causes chloroplasts to shrink. Under the conditions of phosphorylation there is little ATPase activity in chloroplasts. Uncouplers rarely increase and often decrease ATPase activity. However, ATPase activity is enhanced by electron transport and sulfhydryl compounds

Effects of the plastocyanin antagonists KCN and poly-l-lysine on partial reactions in isolated chloroplasts

A very large number of compounds are known to inhibit chloroplast electron transport at or near photosystem II. However, compounds which have been shown to block photosystem I are very few despite the great importance of such inhibitors in analyzing the pathway of photosynthetic electron transport and the sites of phosphorylation associated with it. Hauska et al. [l] demonstrated that an antibody to plastocyanin can be a useful inhibitor but only when applied to finely fragmented chloroplasts. Kimimura and Katoh [2] have recently reported that incubation of chloroplasts in HgCl, can inhibit electron flow at plastocyanin. However, prolonged exposure to HgCl, has a number of deleterious effects on the chloroplasts; moreover it strongly inhibits phosphorylation [3] . This leaves us only two specific photosystem I inhibitors which can be applied to unfragmented chloroplasts, i.e., polylysine (and certain other polycations) of Brand et al. [4] and KCN of Ouitrakul and Izawa [5] . Brand et al. [6] have located the site of polycation inhibition between cytochrome f and P,,,. Izawa and associates (manuscript in preparation) have shown spectrophotometrically that KCN inhibits cytochrome f oxidation. Moreover KCN has been shown to react readily with isolated plastocyanin under the conditions required for effecting electron transport inhibition. Thus both polylysine and KCN appear to block electron transport at the level of

Hill reaction rates and chloroplast fragment size.

Abstract When the rate of ferricyanide reduction is measured in conventional media containing no phosphorylation uncoupler, the Hill reaction activities found in chloroplast fragments are usually higher than in intact chloroplasts. Even in the smallest particles (supernatant after centrifugation at 145 000 × g ) the residual activity is comparable to the activity of the chloroplasts before fragmentation. However, when the uncoupler methylamine is introduced to eliminate an internal electron-flow barrier associated with the phosphorylation mechanism, the greatly stimulated rate of the uncoupled Hill reaction decreases markedly with decreasing particle size. In the smallest particles (presumably equivalent in size to aggregates of a few “quantasomes”) the uncoupled rate is only 5% of the very high rate in the unfragmented chloroplasts. All disruption methods tested (sonication, grinding, detergent action) gave similar results. Apparently the interaction of two opposing effects is responsible for the illusion that very small chloroplast particles are fully active in the Hill reaction: a progressive loss of activity with decreasing size masked by more and more uncoupling which permits ever higher rates in the particles which remain active. Although particles containing one or a few “quantasomes” have little residual activity, the observed relation of particle size to oxygen producing activity may not be meaningful since there seem to be inactivation processes associated with the act of fragmentation. The fragmentation-dependent uncoupling is at least partially explicable in terms of the increased sensitivity of the smaller fragments to the uncoupling action of the anions inevitably present in the buffered medium.