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The charge separation and excited states of antenna bacteriochlorophyll in membrane fragments of the recently discovered photosynthetic bacterium Helobacterium chlorum were studied by absorbance-difference spectroscopy. Formation ofsinglet excited states of bacteriochlorophyll g with a lifetime of 200 ps or less was observed as the disappearance of the ground state absorption bands. From the absorbancedifference spectra, it was concluded that the primary photochemical reaction consists of the transfer of an electron from the primary donor P-798 to a possibly bacteriochlorophyll c-like pigment absorbing at 670 nm. Electron transfer to the secondary acceptor occurred with a time constant of about 500 ps. The midpoint potential of this acceptor (between -450 and -560 mV) and the absence of sigifcant absorbance changes in the near-infrared upon its reduction suggest that this acceptor is an iron-sulfur center. It is concluded that the primary photochemistry of H. chlorum is similar to that of green sulfur bacteria.The recently discovered photosynthetic bacterium Heliobacterium chlorum (1) has an unusual pigment composition and structure. Instead of bacteriochlorophyll (BChl) a or b, it contains BChl g (2) and a pigment absorbing at 670 nm, and it does not appear to possess either chlorosomes, like green bacteria, or invaginations of the cytoplasmic membrane, like purple bacteria. Nothing is known about its mechanism of photosynthetic electron transport, except that illumination results in (I) the bleaching of a pigment absorbing at 798 nm that may serve as primary electron donor and (ih) the oxidation of a c-type cytochrome (3).Recently we have shown that picosecond absorptiondifference spectroscopy of isolated membranes of the green sulfur bacterium Prosthecochloris aestuarii provides information not only on the properties of the excited states generated in the antenna but also on the components involved in the primary charge separation in this bacterium (4). In the present communication we report the results of a picosecond study of membranes of H. chlorum. Evidence will be presented that the primary photochemistry in this bacterium is similar to that in green sulfur bacteria.MATERIALS AND METHODS H. chlorum was grown anaerobically in medium 112 of the American Type Culture Collection (1) containing 2.5 mM ascorbate. Cells were harvested by centrifugation and resuspended in a buffer of pH 8.0 containing 10 mM Tris, 10 mM ascorbate, and 2 mM dithiothreitol. Membrane fragments were prepared by sonication followed by centrifugation for 15 min at 27,000 x g to remove large cell fragments. The preparation was stored in the dark at 50C before use.For measurements of absorption changes under continuous illumination, the samples were used without further additions. The absorbance changes were measured as in ref.5. Suitable interference and absorbance filters were used to select the actinic illumination and to protect the photomultiplier from stray actinic light. The picosecond absorbancedifference measurements wer...

The transfer of excitation energy and the pigment arrangement in isolated chlorosomes of the thermophilic green bacterium Chloroflexus aurantiacus were studied by means of absorption, fluorescence and linear dichroism spectroscopy, both at room temperature and at 4 K. The low temperature absorption spectrum shows bands of the main antenna pigments BChl c and carotenoid, in addition to which bands of BChl a are present at 798 and 613 nm. Fluorescence measurements showed that excitation energy from BChl c and carotenoid is transferred to BChl a, which presumably functions as an intermediate in energy transfer from the chlorosome to the cytoplasmic membrane. Measurements of fluorescence polarization and the use of two different orientation techniques for linear dichroism experiments enabled us to determine the orientation of several transition dipole moments with respect to each other and to the three principal axes of the chlorosome. The Qy transition of BChl a is oriented almost perfectly perpendicular to the long axis of the chlorosome. The Qy transition of BChl c and the γ-carotene transition dipole are almost parallel to each other. They make an angle of about 40° with the long axis and of about 70° with the short axis of the chlorosome; the angle between these transitions and the BChl a Qy transition is close to the magic angle (55°).

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We have studied the pigment arrangement in purified cytoplasmic membranes of the thermophilic green bacterium Chloroflexus aurantiacus. The membranes contain 30-35 antenna bacteriochlorophyll a molecules per reaction center; these are organized in the B808-866 light-harvesting complex, together with carotenoids in a 2:1 molar ratio. Measurements of linear dichroism in a pressed polyacrylamide gel permitted the accurate determination of the orientation of the optical transition dipole moments with respect to the membrane plane. Combination of linear dichroism and low temperature fluorescence polarization data shows that the Qy transitions of the BChl 866 molecules all lie almost perfectly parallel to the membrane plane, but have no preferred orientation within the plane. The BChl 808 Qy transitions make an average angle of about 44° with this plane. This demonstrates that there are clear structural differences between the B808-866 complex of C. aurantiacus and the B800-850 complex of purple bacteria. Excitation energy transfer from carotenoid to BChl a proceeds with about 40% efficiency, while the efficiency of energy transfer from BChl 808 to BChl 866 approaches 100%. From the minimal energy transfer rate between the two spectral forms of BChl a, obtained by analysis of low temperature fluorescence emission spectra, a maximal distance between BChl 808 and BChl 866 of 23 Å was derived.