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Detergent-free reaction centers from Rhodobacter sphaeroides R26 were used to study the solubilization of reaction centers in various detergents and their effects on reaction center photochemistry. 500Z 100 n-octyl-P-D-glucopyranoside or 51 i 5 Triton X-100 molecules were associated with one reaction center. For N,N-alkylamine N-oxide detergents with chain lengths in the range from 8-13 carbon atoms, the number of detergent molecules associated with the reaction centers increased with decreasing chain length. The amount of detergent molecules associated with the reaction centers decreased almost tenfold if the pH was increased from pH 6 to pH 10. The addition of 5 % 1,2,3-heptanetriol to various detergents lowered the detergentheaction center ratio by a factor of two compared to that for the pure detergent. The detergent concentration at which solubilization of the reaction center occurs was close to the critical micelle concentration for all detergents studied. The absorption band at 865 nm of the primary donor in the reaction center shifts to 846nm when detergent was removed from the reaction center; upon resolubilization with various detergents, this band shifts back to 865 nm. In 80-90% of the detergent-free reaction centers, the secondary electron transfer from QA to QB was inhibited; this electron transfer was restored after re-addition of detergent.Keywords: photosynthesis ; membrane protein ; detergent: crystallization; Rhodobacter sphaeroides.Crystallization of detergent-solubilized membrane proteins is complicated by the randomly ordered detergent molecules surrounding the proteins. The success of crystallization of membrane proteins depends on optimizing hydrophobic and hydrophilic interactions between the protein molecules. The type of detergent plays an important role in this process. The length of the detergent alkyl chain determines the thickness of the detergent belt around the protein. This belt should be as thin as possible to increase the number of contact sites between the membrane proteins ; however, the detergent belt should not be too small, so that the detergents can form bridges between the proteins inside the crystal structure [I]. Additionally, the polar head group of the detergent should be small to ensure optimal hydrophilic interactions between the protein molecules [l].The first successful crystallization of a membrane protein was achieved with the photosynthetic reaction center (RC) protein from the purple bacterium Rhodopseudomonas viridis [2]. The addition of 1-3% of the amphiphillic molecule 1,2,3-heptanetriol was essential for the formation of crystals. This led Michel [3] to postulate that the dimensions of the micellar disk surrounding the protein are fine-tuned by 1,2,3-heptanetriol so as to fit into the crystal structure and to increase proteidprotein interactions. Measurements on the micellar radius of N,Ndimethyl dodecylamine N-oxide (DodNOMe, or LDAO) in solution have shown a decrease of the radius upon addition of 1,2,3-heptanetriol The size of this detergent belt is d...

Detergent-free reaction center (RC) proteins from the photosynthetic bacterium Rhodopseudomonas viridis were obtained using Bio-Beads SM-2. With these RCs, the amount of detergent molecules associated with the protein was measured by determining the detergent concentration at which re-solubilization occurred as a function of the RC concentration. For N, N-dimethyl dodecylamine-N-oxide (LDAO), Triton X-100 and j?-octylglucoside 260 + 30,105 + 10 and 360 f 100 detergent molecules were necessary to dissolve the protein, respectively. With this technique we have studied the effect of the amphiphilic molecule 1,2,3-heptanetriol, which is essential in the crystallization process of these RCs. Addition of 5% 1,2,3-heptanetriol reduces the value for LDAO to 120 k 20 LDAO/RC, supporting the notion that crystallization of the RCs is promoted by increasing the number of protein-protein contacts.

Electric field-induced charge recombination in Photosystem II (PS II) was studied in osmotically swollen spinach chloroplasts ('blebs') by measurement of the concomitant chlorophyll luminescence emission (electroluminescence). A pronounced dependence on the redox state of the two-electron gate QB was observed and the earlier failure to detect it is explained. The influence of the QB/QB (-) oscillation on electroluminescence was dependent on the redox state of the oxygen evolving complex; at times around one millisecond after flash illumination a large effect was observed in the states S2 and S3, but not in the state 'S4' (actually Z(+)S3). The presence of the oxidized secondary electron donor, tyrosine Z(+), appeared to prevent expression of the QB/QB (-) effect on electroluminescence, possibly because this effect is primarily due to a shift of the redox equilibrium between Z/Z(+) and the oxygen evolving complex.

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