Don Devault

Low temperature photo-induced reactions in green leaves and chloroplasts

Abstract 1. Swiss chard leaves, spinach leaves and spinach chloroplasts at liquid nitrogen temperatures exhibit photo-induced absorbance changes with maxima at 556–557, 680–682 and 703–705 nm. Experiments reported indicate that the 556 nm peak is due to the α-band of cytochrome b559. The peak at 680 nm is attributed to P680, the photoactive center of photosystem II, and the peak at 703 nm is attributed to P700. 2. Laser-induced oxidation of cytochrome b559 has a half-time of 4.6 msec. Laser oxidized P680 has two reductive phases, one having a half-time of 30 μsec and the other of 4.5 msec. P700 has only one reductive phase with a half-time of approx. 30 μsec. The kinetics strongly suggest that P680 is the primary oxidant of cytochrome b559 at low temperatures. 3. The slow reductive phase of P680 and oxidation of cytochrome b559 tended to have equal and approximately constant rates between 80 and 220°K.

The site of KCN inhibition in the photosynthetic electron transport pathway

Treatment of chloroplasts with high concentrations of KCN inhibits reactions which involve Photosystem I (e.g. electron transport from water or diaminodurene to methylviologen), but not those assumed to by-pass Photosystem I (e.g. electron transport from water to quinonediimides). The spectrophotometric experiments described in this paper showed that KCN inhibits the oxidation of cytochrome f by far-red light without blocking its reduction by red light. Both optical and EPR experiments indicated that KCN does not inhibit the photooxidation of P700 but markedly slows down the subsequent dark decay (reduction). Reduction of P700 by Photosystem II is prevented by KCN. It is concluded that KCN blocks electron transfer between cytochrome f and P700, i.e. the reaction step which is believed to be mediated by plastocyanin. In KCN-poisoned chloroplasts the slow dark reduction of P700 following photooxidation is greatly accelerated by reduced 2,6-dichlorophenolindophenol or by reduced N-methylphenazonium methosulfate (PMS), but not by diaminodurene. It appears that the reduced indophenol dye and reduced PMS are capable of donating electrons directly to P700, at least partially by-passing the KCN block.

Energy transduction in electron transport.

Abstract Mathematical formulae and computations are presented which may be useful in understanding equilibrium phenomena at an energy transduction site in an electron transport chain, such as variation of apparent midpoint potential with phosphate potential, crossover phenomena, and respiratory control. The model used has been published previously without the present calculations and is intended to be the simplest that can be conceived on purely physicochemical principles. Several alternative explanations of the Wilson−Dutton data are given.

Photosynthetic reaction center transients, P435 and P424, in Chromatium D

Abstract 1. The correlation of kinetic, saturation and potentiometric evidence shows that P 435 is a spectral component of the same reaction center as P883 in Chromatium D. 2. There is only one cytochrome oxidizing reaction center P883/P 435 , since the spectrum of P 435 is the same independent of which cytochrome (C 553 or C 555 ) is oxidized and the P 435 reduction kinetics correlate with the oxidation kinetics of either cytochrome, depending upon which is being oxidized. 3. The oxidation half-time of P883/P 435 is less than 50 nsec. 4. The E m of P883/P 435 is +486 mV and that of its primary electron acceptor is −134 mV. 5. P 424 is observable only when the primary electron acceptor, X, is in the reduced state prior to the flash: (A) at room temperature in chromatophores between −318 and −145 mV and (B) at 77°K over the same potential range (first laser flash) or at higher potentials (subsequent laser flashes) under conditions in which the first laser flash permanently reduces X. 6. P424, P883/P 435 , cytochrome C 553 and cytochrome C 555 are all associated with Thornbers subchromatophore Fraction A, but not Fraction B. 7. The rise half-time of P 424 in chromatophores is 50 nsec at room temperature and 200 nsec at 77°K. Its recovery half-time is 2.2 to 2.4 μsec at both temperatures in chromatophores or 16 msec in whole cells at 77°K. 8. P 424 might represent either an oxidation of bacteriochlorophyll spectrally modified by the presence of reduced primary electron acceptor or a light-induced reduction state of bacteriochlorophyll. 9. Thermodynamically, it is possible that this new species might be a link in the direct reduction of NAD + .

Symmetry, orientation and rotational mobility in the a3 heme of cytochrome c oxidase in the inner membrane of mitochondria.

The photoinduced linear dichroism of absorption changes resulting from photolysis of the complex between heme a3 of the cytochrome oxidase and CO is studied. The experiments started from isotropic solutions or suspensions of the enzyme both in its isolated form and in mitochondria. The anisotropy responsible for the linear dichroism was induced by excitation with a flash of linearly polarized light. The dichroic ratios observed with various systems; polymerized enzyme in solution, enzyme in mitochondria and in submitochondrial particles (at 20 degrees C as well as at liquid N2-temperature) all approached a value of 4/3 which characterizes a chromophore which is circularly degenerate. Therefrom we conclude that the interaction of heme a3 with its microenvironment within the protein does not break its four-fold symmetry. The experiments with mitochondria and submitochondrial particles suspended in aqueous buffer revealed similarly high dichoric ratios without any dichroic relaxation other than a rather slow one which could be attributed to the rotation of the whole organelle in the suspending medium. Therefrom we conclude that the cytochrome oxidase either is totally immobilized in the membrane, or that it carries out only limited rotational diffusion around a single axis coinciding with the symmetry axis of heme a3. In the light of independent evidence for a transmembrane arrangement of the oxidase and for the general fluidity of the inner mitochondrial membrane we consider anisotropic mobility of the cytochrome oxidase around an axis normal to the plane of the membrane as the most likely interpretation. Then our experimental results imply that the plane of heme a3 is coplanar to the membrane.

Theory of iron-sulfur center N-2 oxidation and reduction by ATP.

Abstract The data of Ohnishi (1975) and of Gutman and coworkers on iron-sulfur center N-2 in mitochondria and submitochondrial particles are examined in as much quantitative detail as possible from the standpoint of both chemiosmotic theory and of chemical intermediate (transductase) theory. A method of examination of the behavior of an energy transduction site by plotting its properties as a function of both the high and low redox potentials on either side of the site is described in some detail. That adding ATP causes center N-2 to go oxidized when buffered redox-wise on the low potential side and reduced when buffered on the high potential side can be explained by both chemiosmotic and chemical intermediate theory. Chemiosmotic explanations consistent with the data exclude location of N-2 at the inside of the mitochondrial membrane, but location at the out side or the middle or mobile across the membrane cannot be ruled out by present data. All four abridged transductase models of chemical intermediate theory can be fitted to the data by choice of parameters. That center N-2 is a simple redox couple located at either side of energy transduction site 1 is ruled out. Further experiments needed to clarify present ambiguities are shown to be: (i) Adding ATP while buffering (redox-wise) the NAD + -NADH inside whole mitochondria; (ii) mapping the apparent midpoint potential or, alternatively, the redox state as a complete function of the redox potentials on both the high and low sides; (iii) determination of differences that may be caused by sidedness of the preparation (mitochondria or submitochondrial particles); and (iv) determining effects of changing the partitioning of the proton motive force between ΔpH and membrane potential.

EFFECTS OF HIGH PRESSURE ON PHOTOCHEMICAL REACTION CENTERS FROM RHODOPSEUDOMONAS SPHEROIDES

Abstract— Photochemical reaction centers from Rhodopseudomonas spheroides were subjected to pressures ranging from 1 to 6000 atm. Optical absorption, fluorescence and photochemical activities were studied under these conditions.

A low potential photosystem in Chromatium D

A low potential light reaction in chromatophores of Chromatium D has been studied by means of oxidation—reduction potential titrations of laser-induced absorbance changes at 435 nm and 410 nm. This reaction, operating at potentials which are low enough to inactivate the P883 (P435) reaction center, has an apparent Em of −145 mV and is attenuated as the potential is lowered with an Em of −318 mV (n = 1). Its spectrum at −260 mV has a peak at 424 nm (compared with peaks at 423 nm and 432 nm for P435 at +415 mV), and its rise half-time appears to be around 75 nsec (compared with <50 nsec for P435). Although this system may represent either a new reaction center or a different form of P883, it is apparent that thermodynamically it is able to donate electrons to NAD+.

CAROTENOID TRIPLET STATE IN R. SPHEROIDES GA CHROMATOPHORES

It has been suggested by Griffiths et aI (1955) that carotenoids in photosynthetic systems function in part to protect the systems from damage by photooxidation. The mechanism of photooxidation was suggested to be that of energy transfer from the lowest triplet state of chlorophyll a to molecular oxygen (Foote et al., 1970), or to be that of electron transfer from the triplet state of bacteriochlorophyll to oxygen (Connolly et al., 1973). Connolly et al. (1973) have shown that, in uitro, carotenoids, possibly through energy exchange, readily quench the bacteriochlorophyll triplet with formation of carotenoid triplet. Mathis (1969) studied the triplet-triplet energy transfer from chlorophyll a to carotenoids. Witt et al. (1970) have demonstrated the formation of carotenoid triplet in chloroplasts under high light intensity. Cogdell er d. (1975) reported on carotenoid triplet formation in reaction centers isolated from photosynthetic bacteria. In this paper we proceed to study the formation and characteristics of the carotenoid triplet in photosynthetic bacteria in the nanosecond and early microsecond time regions to fill the information gap between the picosecond studies of Leigh et al. (1974) and the late microsecond studies (Jackson et al., 1969, 1973; Baltscheffsky, 1969) on carotenoids of chromatophores.

High-order fluorescence and exciton interaction in photosynthetic bacteria

We have observed fluorescence at visible wavelengths from chromatophores of photosynthetic bacteria excited with infrared radiation which we attribute to bacteriochlorophyll of the antenna system. The fluorescence is prompt (no delay greater than 5 ns). Its spectrum shows peaks at 445, 530 (broad) and 600 nm when excited with either 694 or 868 nm. Quantum yield is of the order of 10(-9). The dependence on intensity indicates generation by mainly third-order processes which could involve triplet state in combination with excited singlets. Second-order single-singlet fusion could also contribute. The high-order fluorescence can also be explained as arising from absorption of a second photon by singlet excited states.