Sphaeroides R26 Reaction Centers Research Articles

Does different orientation of the methoxy groups of ubiquinone-10 in the reaction centre of Rhodobacter sphaeroides cause different binding at QA and QB?

The different roles of ubiquinone-10 (UQ10) at the primary and secondary quinone (QA and QB) binding sites of Rhodobacter sphaeroides R26 reaction centres are governed by the protein microenvironment. The 4C=O carbonyl group of QA is unusually strongly hydrogen-bonded, in contrast to QB. This asymmetric binding seems to determine their different functions. The asymmetric hydrogen-bonding at QA can be caused intrinsically by distortion of the methoxy groups or extrinsically by binding to specific amino-acid side groups. Different X-ray-based structural models show contradictory orientations of the methoxy groups and do not provide a clear picture. To elucidate if distortion of the methoxy groups induces this hydrogen-bonding, their (ring-)C-O vibrations were assigned by use of site-specifically labelled [5-13C]UQ10 and [6-13C]UQ10 reconstituted at either the QA or the QB binding site. Two infrared bands at 1288 cm(-1) and 1264 cm(-1) were assigned to the methoxy vibrations. They did not shift in frequency at either the QA or QB binding sites, as compared with unbound UQ10. As the frequencies of these vibrations and their coupling are sensitive to the conformations of the methoxy groups, different conformations of the C(5) and C(6) methoxy groups at the QA and QB binding sites can now be excluded. Both methoxy groups are oriented out of plane at QA and QB. Therefore, hydrogen-bonding to His M219 combined with electrostatic interactions with the Fe2+ ion seems to determine the strong asymmetric binding of QA.

Photoselection effects in laser flash-induced spin-polarised EPR spectra of the radical pair state [P +Q A−] in Rhodobacter sphaeroides reaction centers

The spin-polarised EPR signal of the radical pair state [P +Q A −] in Zn-substituted Rhodobacter sphaeroides R26 reaction centers demonstrates a strong dependence on the wavelength and polarisation of the excitation light. This dependence is attributed to photoselection by the plane-polarised laser light utilised in the experiments. The model of spin-correlated radical pairs describes the experimental results well if the effect of photoselection is taken into account.

Light-induced structural changes in photosynthetic reaction centres studied by ESEEM of spin-correlated D+QA− radical pairs

Zn-substituted Rhodobacter sphaeroides R26 reaction centres (RCs) frozen in the dark and under illumination exhibit quite different recombination kinetics of the D + Q A − radical pairs [Kleinfeld et al., Biochemistry, 23 (1984) 5780]. We have applied electron spin echo envelope modulation (ESEEM) of the spin-correlated D + Q A − radical pairs to assess a possible light-induced change in the distance between the D and Q A cofactors. The recombination kinetics and the field-swept spin-polarized EPR signal for the two preparations have been monitored by time-resolved EPR spectroscopy. For the samples frozen under illumination, a slight increase in the distance, 0.4±0.2 Å, has been detected.

FTIR spectroscopy shows weak symmetric hydrogen bonding of the Q B carbonyl groups in Rhodobacter sphaeroides R26 reaction centres

The absorption frequencies of the C  O and C  C (neutral state) and of the CO (semiquinone state) stretching vibrations of Q B have been assigned by FTIR spectroscopy, using native and site-specifically 1-, 2-, 3- and 4- 13C-labelled ubiquinone-10 (UQ 10) reconstituted at the Q B binding site of Rhodobacter sphaeroides R26 reaction centres. Besides the main C  O band at 1641 cm −1, two smaller bands are observed at 1664 and 1651 cm −1. The smaller bands at 1664 and 1651 cm −1 agree in frequencies with the 1- and 4-C  O vibrations of unbound UQ 10, showing that a minor fraction is loosely and symmetrically bound to the protein. The larger band at 1641 cm −1 indicates symmetric H-bonding of the 1- and 4-C  O groups for the layer fraction of UQ 10 but much weaker interaction as for the 4-C  O group of Q A The FTIR experiments show that different C  O protein interactions contribute to the factors determining the different functions of UQ 10 at the Q A and the Q B binding sites.

Asymmetric binding of the 1- and 4-C=O groups of QA in Rhodobacter sphaeroides R26 reaction centres monitored by Fourier transform infra-red spectroscopy using site-specific isotopically labelled ubiquinone-10.

Using 1-, 2-, 3- and 4-13C site-specifically labelled ubiquinone-10, reconstituted at the QA site of Rhodobacter sphaeroides R26 reaction centres, the infra-red bands dominated by the 1- and 4-C = O vibration of QA are assigned in the QA(-)-QA difference spectra. The mode dominated by the 4-C = O vibration is drastically downshifted in the reaction centres as compared with its absorption frequency in free ubiquinone-10. In contrast, the mode dominated by the 1-C = O vibration absorbs at similar frequencies in the free and the bound forms. The frequency shift of the 4-C = O vibration is due to a large decrease in bond order and indicates a strong interaction with the protein microenvironment in the ground state. In the charge-separated state the mode dominated by the semiquinone 4-C = O vibration is characteristic of strong hydrogen bonding to the microenvironment, whereas the mode dominated by the 1-C = O vibration indicates a weaker interaction. The asymmetric binding of the 1- and 4-C = O groups to the protein might contribute to the factors governing different redox reactions of ubiquinone-10 at the QA site as compared with its reactions at the QB site.

13C magic angle spinning NMR study of the light-induced and temperature-dependent changes in Rhodobacter sphaeroides R26 reaction centers enriched in [4'-13C]tyrosine.

Solid-state 13C magic angle spinning (MAS) NMR has been used to investigate detergent-solubilized photosynthetic reaction centers of Rhodobacter sphaeroides R26, selectively enriched in [4-13C]-tyrosine. The reaction centers were frozen, in the dark and while subject to intense illumination, and studied at temperatures between approximately 215 and approximately 260 K. The signal consists of at least seven narrow lines superimposed on a broad doublet. The chemical shift anisotropy is similar to that for crystalline tyrosine. The two narrowest resonances, corresponding to signals from individual tyrosines, are 28 +/- 5 Hz wide, comparable to what is observed for quaternary carbons in linearly elastic organic solids. The line width as well as the chemical shift of these signals is essentially independent of temperature. This provides strong evidence for an unusually ordered, well-shielded, and structurally, electrostatically, and thermodynamically stable interior of the protein complex without structural heterogeneities. As the temperature is lowered, additional signal from the labels develops and the natural abundance resonances from the detergent broaden, providing evidence for considerable flexibility at the exterior of the protein complex and in the detergent belt at the higher temperatures. In addition, the NMR provides evidence for an electrostatically uniform and neutral complex, since the total dispersion in isotropic shifts for the labels is < 5 ppm and corresponds to electron density variations of less than 0.03 electronic equivalents with respect to tyrosine in the solid state or in solution. When the sample is frozen while subject to intense illumination, a substantial part of the protein is brought into the charge-separated state P.+QA.-. At least three sharp resonances, including the narrowest lines, are substantially reduced in intensity. It is argued that this effect is caused by the electronic spin density associated with the oxidized primary donor P.+. These results strongly suggest that the environment of the special pair is extremely rigid and question the role of protein conformational distortions during the primary photoprocess.

An assessment of the mechanism of initial electron transfer in bacterial reaction centers

Subpicosecond time-resolved photodichroism measurements on Rhodobacter sphaeroides R26 reaction centers are reported in the key region between 620 and 740 nm, where the anions of both bacteriopheophytin and bacteriochlorophyll (BChl) have their most diagnostic absorption bands. These measurements fail to resolve clearly the formation of a reduced BChl species. The implications of this for elucidating the role of the accessory BChl in the initial stage of charge separation are discussed.

Monomeric bacteriochlorophyll is required for the triplet energy transfer between the primary donor and the carotenoid in photosynthetic bacterial reaction centers

Reaction centers from the carotenoidless mutant Rb. sphaeroides R26 were treated with sodium borohydride which is known to remove one of the accessory monomeric bacteriochlorophylls (BB). Subsequently, the carotenoid, spheroidene, was incorporated into the modified reaction centers. It is demonstrated by optical absorption and circular dichroism experiments that spheroidene, reconstituted into the sodium borohydride-treated Rb. sphaeroides R26 reaction centers, is bound in a single site, in the same environment and with the same structure as spheroidene reconstituted into untreated (native) Rb. sphaeroides R26 reaction centers. Transient optical and electron spin resonance spectroscopic data indicate that unless the accessory BB is present, the primary donor-to-carotenoid triplet energy transfer reaction is inhibited. These observations provide direct evidence for the involvement of the accessory BB in the triplet energy transfer pathway.