Mitsuo Nishimura

Studies on bacterial photophosphorylation. IV. On the maximum amount of delayed photophosphorylation induced by a single flash.

Abstract With a use of a sensitive pH meter, the amount of delayed photophosphorylation in Rhodospirillum rubrum chromatophores was determined. The amount of delayed photophosphorylation was proportional to the irradiant energy of flash in the low-intensity range. The maximum level of the process was attained after the flashes of higher intensities. By flashing illumination of saturating intensity, the amount of delayed photophosphorylation was proportional to the amount of bacteriochlorophyll. The maximal amount of delayed photophosphorylation was determined to be 0.047 ATP per bacteriochlorophyll. The quantum yield and the energy efficiency of the delayed photophosphorylation were discussed. A tentative value of about 2 ATP per electron was calculated as a number of ATP molecules formed per electron transferred in the oxidation-reduction system in the chromatophores.

Studies on the electron-transfer systems in photosynthetic bacteria. II. The effect of heptylhydroxyquinoline-N-oxide and antimycin A on the photosynthetic and respiratory electron-transfer systems.

Heptylhydroxyquinoline-N-oxide and antimycin A were used to study the sequence of oxidation-reduction catalysts in the photosynthetic and respiratory electron-transfer systems of photosynthetic purple bacteria, Rhodopseudomonas spheroides and Rhodospirillum rubium. These inhibitors caused steady-state changes of cytochromes in aerobic-cell suspensions: more reduced cytochrome b and more oxidized cytochrome c. The reduction of cytochrome b (by anaerobiosis) was not inhibited by these reagents but that of cytochrome c was blocked. It was found, too, that these reagents affected the steady-state level of carotenoids in R. spheroides in an anaerobic suspension. Illumination of aerobic cells caused the reduction of cytochrome b and oxidation of cytochrome c in the presence of heptylhydroxyquinoline-N-oxide. It was concluded that these inhibitors block the oxidation-reduction reaction between cytochromes b and c, and that b-type cytochrome occurs nearer to the photochemical reducing site and to dehydrogenases, while c-type is closer to the photochemical and respiratory oxidizing sites.

Studies on bacterial photophosphorylation I. Kinetics of photophosphorylation inRhodospirillum rubrum chromatophores by flashing light

By means of the flashing-light technique, it was shown that photochemical process and dark process can be separated in photophosphorylation ofR. rubrum chromatophores. The first is a rapid process which occurs only during illumination, and this reaction proceeds proportionally to the amount of light given during flash. The second is a slower reaction which occurs both in the dark and during illumination. Owing to the second process, delayed photophosphorylation was observed between flashes. Half-life of decay of the delayed photophosphorylation and the yield of this process per single flash, depend on the light intensity used for flashing illumination.

Light-induced intravesicular pH changes in Rhodospirillum rubrum chromatophores

Abstract The nature of the kinetics of proton movements in subcellular fractions of photosynthetic bacteria can be of importance in determining whether these protons arise primarily from electron transfer reactions or secondarily from high energy chemical “inside” of vesicular chromatophores. Bromthymol blue has been tested in detail intermediates of these electron transfer reactions. In the first case, protons must move in synchrony with electrons; in the second case, no compulsory relationship is involved. More rapid and more sensitive measurements of proton movements can be obtained by optical measurements of absorbance changes of pH indicators bound on the “inside” of vesicular chromatophores. Bromthymol blue has been tested in detail with mitochondria and submitochondrial particles, and has been found to be an “inside” indicator of proton movement. This technique has been applied to suspensions of chromatophores prepared from Rhodospirillum rubrum. At about 4 n-Einsteins/ cm2/second no accelerated proton movement can be detected in the interval that electron transport pigments are approaching their steady state levels ( t 1 2 ~ 50 msec ). In the following 200 msec, proton movements amount to one per 500 chlorophylls, and this rate does not exceed the rate in the following several seconds. The quantum efficiency of hydrogen ion production is low and appears to fall from 0.1 to 0.02 over the range of light intensities employed here. Over this range, the light-induced pigment oxidation is essentially light-limited and its rate is proportional to the light intensity. Thus the rate of proton movement appears to be more closely related to phosphorylation and ion movements in chromatophores than to their electron transport rate. These data afford no experimental evidence for fast hydrogen ion movements compulsorily linked to light-activated electron transport in R. rubrum chromatophores.

Studies on bacterial photophosphorylation II. Effects of reagents and temperature on light-induced and dark phases of photophosphorylation inRhodospirillum rubrum chromatophores

The flashing-light technique in combination with inhibitors and temperature variation was used to study the light-induced and dark phases of photophosphorylation inR. rubrum chromatophores. The first rapid photochemical step occurs only during illumination; it is not affected by methylphenazonium methosulfate. The second slower dark process occurs both during the flash and between flashes. It is further indicated that the dark process of photophosphorylation is separated into two steps; the electron-transfer process (inhibited by 2-n-heptyl-4-hydroxyquinoline-N-oxide and low temperature, and bypassed by methylphenazonium methosulfate) and the process of esterification of phosphate coupled to electron transfer (slightly inhibited by 2,4-dinitrophenol). It is suggested that the rate of phosphorylation under continuous illumination of high intensity is determined by the electron transport at the site of 2-n-heptyl-4-hydroxyquinoline-N-oxide or antimycin A inhibition.

Light-induced electron transfer, internal and external hydrogen ion changes, and phosphorylation in chromatophores of Rhodospirillum rubrum

Abstract Light-induced electron transfer, internal and external hydrogen ion changes (measured by bromthymol blue and glass electrode methods), and phosphorylation in the isolated chromatophores of Rhodospirillum rubrum were studied under phosphorylating and nonphosphorylating conditions. Under nonphosphorylating conditions, hydrogen ion changes saturated at a lower intensity of excitation light than under phosphorylating conditions. The maximum amounts of nonphosphorylating hydrogen ion changes (internal and external) were much smaller than those of phosphorylating changes. Utilization of the high-energy compound or high-energy state by phosphorylation of ADP competed with the utilization of energy by the process which induces internal acidification. The absence of ADP or addition of oligomycin increased the light-induced internal acidification. The process of internal acidification was slower than phosphorylation. Therefore, it cannot precede phosphorylation. Branching of phosphorylation and internal acidification in the energy transduction chain occurs at the stage of high-energy intermediate. The kinetics of the light-induced absorbance change was markedly affected by phosphorylation. Effects of electron-transfer inhibitors, an accelerator, and uncouplers of phosphorylation on the absorbance changes and internal and external hydrogen ion changes were studied. Electron transfer was a prerequisite for the hydrogen ion changes. The processes of hydrogen ion changes and phosphorylation were most closely connected with the dark electron transfer in the time range of 10 −3 -10 −2 second after pulse excitation. On the other hand, no effect of different phosphorylating conditions or reagents was observed in the rapid light-induced phase of electron transfer.

The sizes of the photosynthetic energy-transducing units in purple bacteria determined by single flash yield, titration by antibiotics and carotenoid absorption band shift

Several approaches to find out the size of energy-transducing unit in ion transport and photophosphorylation in chromatophores of photosynthetic bacteria (Chromatium Strain D, Rhodospirillum rubrum and Rhodopseudomonas spheroides) were applied. Single flash yield (A), titration of ion transport by ionophorous antibiotics (B), and titration of the shift of carotenoid absorption band by antibiotics (C) were the methods used. Method A gave the values of 50–60 as chlorophyll/hydrogen ratio for nonphosphorylating H+ gradient formation, and the value of 25 for the phosphorylating H+ change. By Method B, the value of 200–400 was obtained as the molar ratio of chlorophyll to the gradient collapsing agent. By Method C, much larger values (2000–5000) were obtained as the molar ratio of chlorophyll/ionophore, when we measured the effect of valinomycin on the dark recovery rate constants of carotenoid shift. The different biophysical aspects of these “photosynthetic units” are discussed.

Uncoupling and charge transfer in bacterial chromatophores.

Abstract Uncoupling of photophosphorylation in bacterial chromatophores is obtained with NH 4 Cl or K + (Na + ) plus nigericin in the presence of either valinomycin or suitable permeant anions. The experiments suggest that uncoupling is obtained by the simultaneous abolishment of both pH gradient and membrane potential components of the light-induced electrochemical proton gradient, independently of the nature of the primary event in energy coupling. A general discussion of the relationship between uncoupling and the transfer of charge across the membranes of chromatophores, chloroplasts and submitochondrial particles is presented.

Energy-and electron-transfer systems in algal photosynthesis. II. Oxidation-reduction reactions of two cytochromes and interactions of two photochemical systems in red algae.

The functioning of cytochromes f and b in the electron-transfer chain of red algae, Porphyridium and Porphyra, was studied. Effects of inhibitors, temperature, etc. on the oxidation-reduction reactions of these cytochromes are shown. The interaction of two photochemical systems through dark electron-transfer in the 10-msec range was demonstrated. The reaction time of electron-transfer reactions was measured with pulsed excitation. Two electron-transfer paths, a non-cyclic path connecting two photochemical systems and a cyclic electron-transfer path around photochemical system I are postulated and a scheme for photosynthetic electron transfer in Porphyridium and Porphyra is presented.

Studies on bacterial photophosphorylation. V. Stoichiometry and kinetics of photophosphorylation and adenosine-5'-triphosphatase and effects of divalent metal ions.

Abstract The stoichiometry and kinetics of photophosphorylation and dark ATPase in Rhodospirillum rubrum chromatophores were studied by the methods of pH-change recording, phosphate analysis and chromatography. A stoichiometric ratio of ΔATP/−ΔH+/added ADP = 1.0/0.9/1.0 was observed in photophosphorylation. The presence of Mg2+, ADP and phosphate is necessary for the maximal phosphorylative activity. The rate of photophosphorylation increased remarkably with higher concentrations of these components. The apparent Michaelis constant for ADP was calculated to be 1.5·10−5M, that for phosphate was 1.1·10−4M. The effects of seven species of divalent metal ions on photophosphorylation were compared. Mg2+ had the greatest effect on the activity. Mn2+, Sr2+ and Ca2+ were effective to a lesser extent in certain concentration ranges. Zn2+ was slightly inhibitory and Be2+ and Cd2+ were strong inhibitors. ATPase reaction of chromatophores in the dark was observed. No adenylate kinase activity was detected. Mg2+ was stimulatory for this ATPase reaction. The apparent Michaelis constant for ATP was 1.5·10−4M.