The reaction center (RC) of photosynthetic bacteria is a membrane protein complex that promotes a light-induced charge separation during the primary process of photosynthesis. In the photosynthetic electron transfer chain, the soluble electron carrier proteins transport electrons to the RC and reduce the photo-oxidized special-pair of bacteriochlorophyll. The high-potential iron-sulfur protein (HiPIP) is known to serve as an electron donor to the RC in some species, where the c-type cytochrome subunit, the peripheral subunit of the RC, directly accepts electrons from the HiPIP. Here we report the crystal structures of the RC and the HiPIP from Thermochromatium (Tch.) tepidum, at 2.2-Å and 1.5-Å resolution, respectively. Tch. tepidum can grow at the highest temperature of all known purple bacteria, and the Tch. tepidum RC shows some degree of stability to high temperature. Comparison with the RCs of mesophiles, such as Blastochloris viridis, has shown that the Tch. tepidum RC possesses more Arg residues at the membrane surface, which might contribute to the stability of this membrane protein. The RC and the HiPIP both possess hydrophobic patches on their respective surfaces, and the HiPIP is expected to interact with the cytochrome subunit by hydrophobic interactions near the heme-1, the most distal heme to the special-pair. In photosynthetic purple bacteria, the electron transfer reactions of photosynthesis are performed by the following three components: the photosynthetic reaction center (RC), the cytochrome (Cyt) bc 1 complex, and the soluble electron carrier protein. First, the RC promotes the light-induced charge separation across the plasma membrane, which results in the oxidation of the special-pair and the reduction of the quinone to the quinol. The quinol then leaves the RC and moves to the Cyt bc 1 complex through the quinone pool of the plasma membrane. Second, the Cyt bc 1 complex reoxidizes the quinol to the quinone, and the released electrons are transferred to the soluble electron carriers. Third, the soluble electron carriers transport the electrons to the RC through the periplasmic space. Finally, the photo-oxidized special-pair is reduced by the soluble electron carriers, and the RC comes back to the initial state. In the course of the oxidation and the reduction of the quinones, the transmembrane electrochemical gradient of the protons is formed, and its energy is used to produce ATP by ATP synthase.Thermochromatium (Tch.; formerly Chromatium) tepidum is a purple sulfur bacterium originally isolated from the hot springs in Yellowstone National Park (1, 2) and belongs to the ␥-subclass. Tch. tepidum is a thermophilic bacterium and can grow at the highest temperature of all known purple bacteria. The optimum growth temperature is 48-50°C, the maximum temperature 58°C. The RC from Tch. tepidum is stable up to 70°C in chromatophore and to 48°C in detergent-micelle (3). The RC is the first membrane protein whose three-dimensional structure has been determined at an atomic resolution (4, 5), and the...

We have used near-infrared Fourier transform (pre)resonance Raman spectroscopy to determine the protein interactions with the bacteriochlorophyll (BChl) dimer constituting the primary electron donor, P, in the reaction center (RC) from the thermophilic purple sulfur bacterium Chromatium tepidum. In addition, we report the alignment of partial sequences of the L and M protein subunits of C. tepidum RCs in the vicinity of the primary donor with those of Rhodobacter sphaeroides and Rhodopseudomonas viridis. Taken together, these results enable us to propose the hydrogen-bonding pattern and the H-bond donors to the conjugated carbonyl groups of P. Selective excitation (1064-nm laser radiation) of the FT (pre)-resonance Raman spectra of P in its neutral (P degree) and oxidized (P degree +) states were obtained via their electronic absorption bands at 876 and 1240 nm, respectively. The P degree spectrum exhibits vibrational frequencies at 1608, 1616, 1633, and 1697 cm-1 which bleach upon P oxidation. The P degree + spectrum exhibits new bands at 1600, 1639, and 1719 cm-1. The 1608-cm-1 band, which downshifts to 1600 cm-1 upon oxidation, is assigned to a CaCm methine bridge stretching mode of the P dimer, indicating that each BChl molecule possesses a single axial ligand (His L181 and His M201, from the sequence alignment). The 1616- and 1633-cm-1 bands correspond to two H-bonded pi-conjugated acetyl carbonyl groups of each BChl molecule. with different H-bond strengths: the 1616-cm-1 band is assigned to the PL C2 acetyl group which is H-bonded to a histidine residue (His L176), while the 1633-cm-1 band is assigned to the PM C2 acetyl carbonyl, H-bonded to a tyrosine residue (Tyr M196). Both PL and PM C9 keto carbonyls are free from interactions and vibrate at the same frequency (1697 cm-1). Thus, the H-bond pattern of the primary donor of C. tepidum differs from that of Rb. sphaeroides in the extra H-bond to the PM C2 acetyl carbonyl group; that of PL is H-bonded to a histidine residue in both primary donors (His L168 in Rb. sphaeroides and His L176 in C. tepidum). The P degree/P degree + redox midpoint potentials were measured to be +497 and +526 mV for isolated C. tepidum RCs with and without the associated tetraheme cytochrome c subunit, respectively, and +502 mV for intracytoplasmic membranes. The positive charge localization was estimated to be 69% in favor of PL, indicating a more delocalized situation over the primary donor of C. tepidum than that of Rb. sphaeroides (estimated to be 80% on PL). These differences in physicochemical properties are discussed with respect to the proposed structural model for the microenvironment of the primary donor of C. tepidum.

4RIKEN Harima Institute/SPring-8, Mikazuki-cho, Sayo-gun, Hyogo, JapanThe photosynthetic reaction center (RC) is a transmembrane protein complex that catalyzes light-driven electron transport across the photosynthetic membrane. The complete amino-acid sequence of the H subunit of the RC from a thermophilic purple sulfur bacterium, Thermochromatium tepidum, has been determined for the first time among purple sulfur bacteria. The H subunit consists of 259 amino acids and has a molecular mass of 28 187. The deduced amino-acid sequences of this H subunit showed a significant (40%) degree of identity with those from mesophilic purple nonsulfur bacteria.The determined primary structure of the H subunit was compared with the structures of mesophilic B. viridis and R. sphaeroides based on the three-dimensional structure of the H subunit from T. tepidum, which has been recently determined by X-ray crystallography. One lipid molecule was found in the crystal structure of the T. tepidum RC, and the head group of the lipid appears to be stabilized by the electrostatic interactions with the conserved basic residues in the H subunit. The above comparison has suggested the existence of a lipid-binding site on the molecular surface at which a lipid molecule can interact with the RC in a specific manner.Keywords: H subunit; membrane protein; thermochromatium; puhA; Q B site.The photosynthetic apparatus of purple bacteria consists of a transmembrane protein complex, a photosynthetic reaction center (RC) that catalyzes the light-driven electron transport across the photosynthetic membrane, and surrounding light-harvesting antenna systems (LH1, LH2) that efficiently harvest light energy and funnel this energy to the RC. The RC complexes in most purple bacteria are composed of three polypeptide subunits termed L (light), M (medium), and H (heavy). In addition, in some species, the RC includes a tightly bound c-type cytochrome subunit, that reduces the photo-oxidized primary electron donor.Studies of the structural genes of the RC and the antenna system have shown that the genes encoding the L, M, and cytochrome subunits of the RC (pufL, pufM and pufC) and the genes encoding the a and b subunits of the lightharvesting complex 1 (LH1) (pufA and pufB) are located in the puf operon. In contrast, only the structural gene encoding the H subunit (puhA) of the RC is located in the puh operon, which is approximately 30±40 kb upstream from the puf operon [1].Although the RCs of many species have been extensively studied, the functional role of the H subunit has not yet been well defined. The removal of the H subunit from purified RCs does not affect their activity, and some mutagenesis studies have also indicated that this subunit is not absolutely essential for the primary photochemical activity [2]. In contrast, a recent study has provided evidence that the H subunit is required as a foundation protein for the assembly of the RC and intracytoplasmic membrane [3]. Although the gene encoding the H subunit of the photosynthetic RC has been det...

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