Biochemistry zyxwvu 1990, 29, zyxwvu 11079-1 1088 11079 Reaction Center Photochemistry of Heliobacterium chlorumt Wolfgang Nitschke,* Pierre Sitif, Ursula Liebl, Ute Feiler, and A. William Rutherford Service de Biophysique, Dgpartement de Biologie, CEN Saclay, 91 191 Gif-sur- Yvette Cedex, France, and Max-Planck-Institut fur Biophysik, Heinrich-Hoffmann Strasse zyxwvu 7, 6000 Frankfurt am Main 71, FRG Received June 18, 1990; Revised Manuscript Received August 27, 1990 ABSTRACT: Reaction center photochemistry in Heliobacterium chlorum has been investigated by using EPR and flash absorption spectroscopy at low temperatures. The following results were obtained. At 5 K, in the presence of ascorbate, continuous illumination resulted in the formation of PTg8+ and a reduced ironsulfur center designated FB (g, zyxwvutsrq = 2.07, zyxwvut gv = 1.93, g, = 1.89). This state was stable at low temperatures, but the yield for this reaction was low, and it was estimated that it occurred only in about 3% of the centers upon the first flash. After continuous illumination of a dilute sample for 10 min, still only half of the centers attained this state. In most centers, flash excitation at 5 K produced a state which recombined with time constants of 2.5 ms (-80%) and 850 ps (-20%). These two phases were differently influenced by the redox state of the reaction center, indicating that two different acceptors were involved in the recombination reactions. When continuous illumination was given at 200 K, a second center, designated FA, was additionally reduced (g, = 2.05, g = 1.95, g, = 1.90). High concentrations of dithionite resulted in the chemical reduction of zyxwvutsrq FB and of most oPF,; illumination at 200 K resulted in the further reduction of FA. Two triplet states were identified by EPR and optical spectroscopy. The amplitude of the narrower triplet (101 = 226 zyx X lo4 cm-I) varied with the redox state of the iron-sulfur centers and was influenced by a component thought to be a quinone undergoing double reduction. It correlated with a triplet state observed by flash absorption spectroscopy showing a bleaching at 798 nm and is attributed to a triplet state formed by charge recombination in the reaction center. Its narrowness is taken as an indication of its origin on a pair of bacteriochlorophylls, and its orientation indicates an orientation of the chlorophyll ring plane perpendicular to the membrane plane. The second triplet had a wider splitting (101 = 242 X lod4cm-l), did not vary systematically with redox conditions, corresponds to an optical spectrum with a maximum at 812 nm, and is not ordered in the membrane. It was thus attributed to a triplet located on a BChl g monomer in the antenna. The reaction center photochemistry in H. chlorum is comparable in many respects to that of photosystem I and green sulfur bacteria. Earlier contrasting conclusions are discussed and rationalized in light of the present results. Until about 8 years ago, the reaction centers of bacterial photosynthesis could be classed into two types. First, purple bacteria and ChlorofZexus aurantiacus, a green non-sulfur bacterium, contain a type of reaction center which is well characterized. The second type is that of the green sulfur bacteria, many basic details of which have only recently be- come apparent (Nitschke et al., 1990). In a sense, cyano- bacteria can be viewed as containing both types of reaction centers, in that photosystem I1 can be regarded as a more sophisticated form of a purple bacterial reaction center (RC)' (Rutherford, 1989) whereas photosystem I seems to be an only slightly modified green sulfur bacterial RC (Nitschke et al., 1990). With the discovery of the Heliobacteriaceae,a new photo- synthetic actor entered the scene. The first species to be described was Heliobacterium chlorum [Gest & Favinger, 1983; see also Barber (1985)], followed by the isolation of Heliobacillus mobilis (Beer-Romero & Gest, 1987), Helio- bacterium gestii, and Heliobacterium fasciculum (Ormerod et al., 1990). All members of the family of Heliobacteriaceae are characterized by the possession of BChl g, a hitherto unknown form of bacteriochlorophyll, which seems to be the major pigment in these organisms. 'Supported by the CNRS (URA 1290). W.N. was supported by the Deutsche Forschungsgemeinschaft (DFG), and U.F. was supported by the Leibniz-Programm (to H. Michel) of the DFG and the Max- Planck-Gesellschaft. *Address correspondence to this author at CEN Saclay. The chemical nature of BChl g has been elucidated by Brockmann and Lipinski (1983) and subsequently confirmed by Michalski et al. (1987). According to phylogenetic trees based on 16s RNA com- parisons, Heliobacteria cannot be classed with any of the other known photosynthetic organisms but rather seem to be more closely related to the (nonphotosynthetic) Gram-positive bacteria (Woese, 1987). Since the discovery of Heliobacterium chlorum, research has focused mainly on the reaction center, and EPR, optical spectroscopy, and biochemical methods have been employed (Prince et al., 1985; Fuller et al., 1985; Smit et al., 1987, 1989; Brok et al., 1986; Vos et al., 1989; Trost & Blankenship, 1989; Van de Meent et al., 1990; Fischer, 1990). The midpoint potential of the primary donor was determined to be in the region of +225 to +250 mV (Prince et al., 1985; Brok et al., 1986), i.e., close to that found in green bacteria (Fowler et al., 1971; Prince & Olson, 1976; Knaff et al., 1979). For the terminal acceptor, a midpoint potential of -510 mV has been reported (Prince et al., 1985), which is much lower than that of purple bacteria but comes close to what is found in pho- tosystem I (Golbeck, 1987) and green sulfur bacteria (Knaff et al., 1979; Nitschke et al., 1990). A relationship with re- action centers from green sulfur bacteria has therefore been Abbreviations: RC, reaction center complex; BChl, bacterio- chlorophyll; FeS center, ironsulfur center; PT9*. primary electron donor bacteriochlorophylls of H. chlorum; PS I, photosystem I; PS 11, photo- system 11; kDa, kilodalton(s). 0006-2960/90/0429- 1 1079$02.50/0 0 1990 American Chemical Society