Crystallization of reaction center from Rhodopseudomonas sphaeroides: preliminary characterization.

Abstract

Reaction centers (RCs), integral membrane proteins that mediate the conversion of light into chemical energy, were crystallized by two different vapor diffusion techniques. In one method, small amphipathic molecules (1,2,3-heptanetriol and triethylammonium phosphate) were added to the RCs that had been solubilized in detergent. In the second method, crystallization occurred near the phase boundaries of a two-phase system created by the addition of polyethylene glycol and NaCl to RCs in ocyl /3-D-glucoside. Several different crystal forms were obtained; two were analyzed by x-ray diffraction. One was monoclinic (space group P2) with fi = 105°, and a = 70 A, b = 105 A, and c = 85 A, two RCs per unit cell, and one RC per asymmetric unit; the crystal diffracted to 3.5 A at 17TC. The other crystal form was orthorhombic (space group C222) with a = 185 A, b = 170 A, and c = 105 A, with eight RCs per unit cell and one RC per asymmetric unit. Reversible light-induced EPR signals of the primary donor (bacteriochlorophyll dimer) showed that the RCs in the crystal were fully active. From the angular dependence of the EPR signal the molecular g anisotropy of the bacteriochlorophyll dimer was deduced to be go1-= (64 -+-3) X 10-5. Linear dichroism measurements were performed on the monoclinic crystal. The two bands at 535 and 544 nm assigned to the Q1 transitions of the bacteriopheophytins were resolved and preliminary orientations of some of the pigments were obtained.The reaction center (RC) is an integral membrane protein that mediates the conversion of electromagnetic energy (light) into chemical energy. The RC from the purple bacterium Rhodopseudomonas sphaeroides is composed of three polypeptide chains designated L, M, and H present in a 1:1:1 stoichiometry with a total molecular weight of -100,000. The following cofactors (reactants) involved in the energy transfer process are attached to the RC: four bacteriochlorophylls (BChl), two bacteriopheophytins (BPhe), two ubiquinones, and one nonheme iron (for review see ref. 1).Although a great deal of work has been done on the characterization of RCs, there is little information available about their three-dimensional structure. The lack of knowledge of the exact spatial relationships between the reactants makes it difficult to understand quantitatively the details of the energy transfer process, in particular the remarkably high quantum efficiency of the photosynthetic process. Accordingly, we have begun structural studies on crystals of RCs from R. sphaeroides.Recently several membrane proteins have been crystallized (2-4), including RCs from the photosynthetic bacterium Rhodopseudomonas viridis (5), and some structural studies have been performed (6, 7). However, since RCs from R. sphaeroides are smaller and have been characterized in more detail than those from R. viridis, studies on R. sphaeroides may be easier to interpret. In this work we describe methods of obtaining crystals from R. sphaeroides and present preliminary result of x-ray diffraction, electron pa...

Cloning and expression of the Rhodobacter sphaeroides reaction center H gene

“…In addition, when immune IgG was incubated with the in vitro products from a pRHB2-programmed reaction mixture, a polypeptide which comigrated with the RC-H subunit was precipitated which was 1 In vivo expression of the puhA gene. We analyzed transcription of the puhA gene by Northern hybridization with an internal puhA-specific probe against bulk R. sphaeroides RNA from cells grown either aerobically in a 30% oxygen atmosphere or photosynthetically (Fig.…”

“…Numerous physical and biochemical techniques have been used to study the structure, function, and topography of the RC complex within the membrane, the binding and organization of the photopigment molecules within the RC complex, and the associations of individual RC proteins with each other (15,28,29). The recent preparation and analysis of crystals of purified RC complexes from Rhodopseudomonas virdis (9,10) and R. sphaeroides (1) show the potential for elucidating the detailed structure, organization, and functions of the various components of this complex.…”

FOURIER TRANSFORM INFRARED SPECTROELECTROCHEMISTRY OF THE BACTERIOCHLOROPHYLL a ANION RADICAL

“…Photosynthetic charge separation and stabilization involve the generation of a radical cation at the primary electron donor side and of radical anions at the various steps of the electron acceptor side. These processes take place at the reaction center, a unit best characterized both functionally (for a review, see Parson, 1985) and structurally (Deisenhofer et al, 1984;Chang et al, 1985;Allen and Feher, 1984) for the purple photosynthetic bacteria containing either bacteriochlorophyll a or b (BChl a, BChl b)t in dimeric form as an electron donor as well as their corresponding bacteriopheophytins a or b (BPheo a , BPheo 6) as a first electron acceptor. The identity of these pigments, their chemical nature and their reactions have been characterized by fast optical spectroscopy (Martin et al, 1986;Woodbury et al, 1985;Wasielewski and Tiede, 1986), EPR spectroscopy (Fajer et al.…”

Direct observation of vibrational coherence in bacterial reaction centers using femtosecond absorption spectroscopy.

“…The reaction center of purple bacteria, which are used in this study, normally contains four bacteriochlorophylls [two of which display strong dimeric interaction and are designated P (for "special pair") and two of which, BL and BM, are more monomer-like], two bacteriopheophytins (HL and HM), and two quinones (QA and QB) bound to the protein subunits L and M (8,9). The lower-exciton excited state of the dimer, P*, is the precursor of electron transport; the chargeseparated state P+H-is formed in 0.7-3.5 ps, depending on the species and temperature (10)(11)(12)(13)(14)(15)(16).…”