Chromatium Chromatophores Research Articles

Light-induced electron transfer in Chromatium strain D III. Photophosphorylation by Chromatium chromatophores

Photophosphorylation at relatively high rates occurs in light-particle preparations described previously as derived from Chromatium chromatophores. It is shown that the optimal conditions for photophosphyorylation correlate with changes in absorbance established in previous researches as the result of ambient redox potentials between 50 and 100 mV. Effects of various dyes, phenazine methosulfate, oxygen and ascorbate are presented and discussed. Attempts to demonstrate by direct measurement the effective redox potentials for photophosphorylation are described. The evidence accumulated favors occurrence of a cyclic phosphorylation mechanism associated with one of the electron transport pathways (the ‘P-890 pathway’) postulated in a model presented in a previous paper.

Light-induced electron transport in Chromatium strain D I. Isolation and characterization of Chromatium chromatophores

Fractionation of Chromatium Strain D chromatophores by centrifugation in sucrose density gradients results in two fractions, designated ‘light’ and ‘heavy’. The light particles are enriched in photosynthetic pigments, are relatively homogeneous and can be prepared reproducibly. They do not scatter light appreciably, contain 41% of the bacteriochlorophyll, 26% of the carotenoid, 25% of the heme and 4% of the protein found in intact cells, and are approx. 75% lipid, and 25% protein, 30% of which can be ascribed to cytochromes. The molecular weight is 12.9.10 6. In contrast, the heavy particles are heterogeneous, can not be prepared reproducibly, scatter light and contain approx. 14% of the bacteriochlorophyll, 9% of the carotenoid, 8% of the heme and 6% of the protein present in intact cells. Three meso-heme proteins were identified in the light particles: cytochrome c−552, cytochrome cc′, and cytochrome c−555. Both cytochrome c−552 and cytochrome cc′ bound CO when solubilized but not when bound in the light particles. No protoheme ( b- type cytochrome) was found, even in whole cells. These results permit characterization of the chemical and physical properties of a subcellular fraction of Chromatium Strain D, which does not appear to be an artifact of preparation.

The role of P870 in bacterial photosynthesis

In Chromatium chromatophores, a 30-nsec flash causes an absorbance decrease at 882 nm which is interpreted as an oxidation of P870. The quantum yield is close to one. The reaction is complete in less than 0.5 μsec. It does not occur in the presence of dithionite. With a half-time of 2 μsec, the 882-nm absorbance increases again. Separate measurements show that cytochrome C422 becomes oxidized at the same rate. The 882-nm absorbance increase is interpreted as a reduction of oxidized P870 (P870 +). There appears to be a direct electron transfer between cytochrome C422 and P870 +. The 2-μsec P870 + reduction is generally incomplete at 298 °M. It does not occur at 80 °K, nor does the cytochrome oxidation occur at this temperature. At 298 °K, the addition of N-methylphenazonium methosulfate (PMS) causes the 2-μsec reduction to be complete. In the presence of PMS, the cytochrome oxidation occurs with a quantum yield near one, and its rate and extent are similar to those which occur in whole cells. In Rhodospirillum rubrum chromatophores, the P870 + reduction is much slower. In this species, the initial absorbance changes of P800 are complete within 0.2 μsec. The results support the view that P870 oxidation is the primary chemical reaction of photosynthesis in Chromatium chromatophores.

Pigment-protein complexes derived from Rhodospirillum rubrum chromatophores by enzymatic digestion

Rhodospirillum rubrum chromatophores are acted upon by pancreatin (a crude mixture of pancreatic digestive enzymes) or α-chymotrypsin in the presence of 0.5% Triton X-100 to produce three clearly defined pigment-protein complexes which are separable by sucrose density-gradient centrifugation. The first to appear temporally is a brown band which contains carotenoids in an unusual combination, since the complex does not show the absorption peaks in the 400 to 500 nm range normally shown by carotenoids in the chromatophore. The complex does show a large absorption band with a maximum at 368 nm. The addition of acetone and methanol to this complex allows the usual absorption properties of the carotenoids to appear. Two bacterio-chlorophyll (Bchl)-protein complexes are formed. A blue Bchl-protein complex has absorption maxima at 922, 835, 580 and 372 nm. The 922 nm band is partially bleached by addition of ferricyanide, indicating there are two Bchl types in the complex. Addition of ascorbate largely restores the 922 nm peak. The band at 372 nm is due to some carotenoids which are present. A green Bchl-protein complex has absorption maxima at 780, 587 and 360 nm. The green complex (but not the blue) is active in simple photochemical electron transfer reactions such as reduced phenazine metho-sulfate oxidation in the presence of ubiquinone and the photoreduction of 5,5′-dithiobis-(2-nitrobenzoic acid) in the presence of ascorbate and 2,6-dichlorophenolindo-phenol. The green complex is more labile than the blue complex once they are formed. The Bchl-protein complexes could represent modified Bchl-protein complexes which exist in situ or could be formed from Bchl and proteins which are liberated by the digestive enzymes. Treatment with α-chymotrypsin or pancreatin produces similar complexes from R. rubrum chromatophores. Treatment of a blue-green mutant of R. rubrum which lacks carotenoids yields the blue and the green bands, but shows no brown band following centrifugation. Treatment of Chromatium chromatophores with either pancreatin or α-chymotrypsin in the presence of Triton X-100 results in complete digestion of the chromatophore with no discernible pigment complexes being formed.

Nicotinamide adenine dinucleotide photoreduction with Chromatium and Rhodospirillum rubrum chromatophores

The photoreduction of nicotinamide adenine dinucleotide (NAD) catalyzed by Rhodospirillum rubrum and Chromatium chromatophores was compared. Both preparations catalyze a light-induced electron transfer from either succinate or reduced 2,6-dichlorophenolindophenol (DPIPH 2) to NAD. Varying concentrations of quinacrine, oligomycin, and gramicidin inhibited the photoreductions in a similar manner, regardless of which electron donor or chromatophore preparation was employed. The light-induced transfer of electrons from DPIPH 2 (reduced by excess ascorbate) to NAD was inhibited by 2- n-heptyl-4-hydroxyquinoline-N-oxide (HOQNO) in the case of Chromatium chromatophores, but not with R. rubrum chromatophores. Rhodospirillum rubrum particles were equally sensitive to carbonylcyanide m-chlorophenyl-hydrazone ( m-Cl-CCP) inhibition, regardless of which electron donor was employed. In contrast, electron transfer from DPIPH 2 to NAD in Chromatium preparations was more sensitive to m-Cl-CCP than the transfer from succinate to NAD. Inhibition of photophosphorylation with Chromatium preparations by m-Cl-CCP, HOQNO and o-phenanthroline paralleled the inhibition of the photo-induced reduction of NAD by succinate. At a given concentration of inhibitor photophosphorylation was more sensitive to oligomycin and less sensitive to gramicidin than NAD photoreduction. Reduced ubiquinone-2 also serves as an electron donor for the Chromatium system.

Studies on bacterial chromatophores. II. Energy transfer and photooxidative bleaching of bacteriochlorophyll in relation to structure in normal and carotenoid-depleted Chromatium.

Electronic energy transfer, fluorescence emission spectra and photooxidative bleaching of bacteriochlorophyll in Chromatium chromatophores were shown to be affected by detergent action and by inhibition of normal carotenoid synthesis in the parent cells. These phenomena are discussed in relation to the structure of the chromatophore and the state of bacteriochlorophyll in vivo. A structural role for carotenoids is suggested.

PRIMARY REACTIONS IN BACTERIAL PHOTOSYNTHESIS—III. REACTIONS OF CAROTENOIDS AND CYTOCHROMES IN ILLUMINATED BACTERIAL CHROMATOPHORES

SummaryWashed or air‐dried chromatophores of photosynthetic bacteria show a reversible light‐induced bleaching and red shift of the absorption bands of carotenoids. This change is sensitized by light absorbed by bacteriochlorophyll (BChl). The carotenoid reaction is abolished by exposure to deoxycholate. In dried preparations, the reaction is optimal in the presence of an extremely low water vapor concentration. All of the light‐induced absorbancy changes in chromatophores are suppressed reversibly by exhaustive desiccation.Deoxycholate treatment and drying do not affect the efficiency of energy transfer from carotenoids to BChl in Rhodopseudomonas spheroides chromatophores, nor do these treatments affect the way in which carotenoids modify the absorption spectrum of BChl.The carotenoid reaction in washed or dried chromatophores is interpreted as arising from the diffusion of electrons or holes from the neighborhood of a special BChl component (BChl,) to the carotenoid molecules.Dark‐adapted Chromatium chromatophores show, in addition to the light‐induced alteration of BChl2 the reversible light‐induced oxidation of a cytochrome. Correspondingly, R. spheroides chromatophores show the oxidation of cytochrome c553. The cytochrome reaction occurs reversibly in dried films of Chromatium chromatophores; it is absent in dried chromatophores of R. spheroides.In Chromatium the reactions of cytochrome and BChl, show a close kinetic relationship. It is inferred that BChl, is a photosynthetic reaction center that traps excitation energy and initiates the electron‐transfer processes of photosynthesis.

PRIMARY REACTIONS IN BACTERIAL PHOTOSYNTHESIS—I. THE NATURE OF LIGHT‐INDUCED ABSORBANCY CHANGES IN CHROMATOPHORES; EVIDENCE FOR A SPECIAL BACTERIOCHLOROPHYLL COMPONENT

SummaryLight‐induced optical changes have been examined in chromatophores of Rhodopseudonionas spheroides (wild type and blue‐green mutants), Rhodospirillum rubrum and Chromatium. The bacteriochlorophyll (BChl) types absorbing at 850 and 870–890 mp in these chromatophores could be separated by exposure to deoxycholate, followed by differential centrifugation. By the use of this treatment and the choice of various phenotypes preparations were obtained showing great variety in the relative heights of the BChl absorption maxima at 800, 850 and 870–890 mµ.The light‐induced changes included a bleaching of the long‐wave (870–890 mk) band and blue shifts of the BChl absorption bands at 375, 590 and 800 mp. In addition, new absorption bands appeared at 420–450, 980, 1140 and 1250 mp. All these effects except the blue shift at 590 mp (and perhaps also at 375 mp) were duplicated by mild oxidation (470 mV). The interpretation is made that the loss of a ground‐state electron in BChl eliminates one optical transition (bleaching at 870–890 mp) and perturbs the energy levels of other transitions (blue shifts).The spectrum of changes induced by light or mild oxidation remains constant while the absorption spectrum varies widely from one preparation to another; it is concluded that the changes reflect alteration of a special BChl component (BChl,) comprising 2–5 per cent of the total BChl. This conclusion is reinforced by the finding, to be reported in the third paper of this series(8), that the reaction of BChl, is related kinetically to the light‐induced oxidation of a cytochrome. It is proposed that BChl, may act as an energy sink and an agent for stabilizing excitation energy in the form of separated charge.

Absorption spectra of bacterial chromatophores at temperatures from 300° K to I° K

Absorption spectra of chromatophores from R. spheroides and from Chromatium have been measured at temperatures from 300° K to 1.3° K. The absorption bands of bacteriochlorophyll are narrowed and intensified slightly, but not dramatically, at low temperature. The spectrum of R. spheroides chromatophores is not changed by drying them; cooling the dried material changes the infra-red absirption bands of bacteriochlorophyll in a manner suggesting a suppression of bacteriochlorophyll-carotenoid interactions. Such a change is produced in Chromatium chromatophores simply by drying them; the change becomes greater as the dried Chromatium chromatophores are cooled.

Relation of photosynthetic activity to carotenoid-bacteriochlorophyll interaction in Chromatium

Evidence has been presented that the ability of the carotenoids to transfer energy to bacteriochlorophyll for photosynthetic phosphorylation by Chromatium chromatophores depends upon the composition of the isolation medium. Inactivation of the carotenoids is associated with changes in the infrared absorption spectrum of bacteriochlorophyll which resemble the effects of carotenoid deficiency.

Studies on bacterial chromatophores I. Reversible disturbance of transfer of electronic excitation energy between bacteriochlorophyll-types in chromatium

Desocycholate affects the transfer of excitation energy between bacteriochlorophyll-types in Chromatium chromatophores. It is suggested, that the detergent fragments the chromatophore into subunits and subsequenyly disturbs the spatial arrangement of these bacteriochlorophyll-types.