Structural Asymmetry of Bacterial Reaction Centers: A Q_y Resonant Raman Study of the Monomer Bacteriochlorophylls

Abstract

To distinguish contributions from either of the monomer bacteriochlorophyll cofactors BL and BM, we have recorded Raman spectra of reaction centers (RCs) from Rhodobacter sphaeroides, strain R26.1 at low temperature and at resonance with the Q y electronic band at 800 nm. Spectra excited from ferricyanide-treated RCs at 800 nm involved a single BChl species, that we identify with the BL cofactor. Spectra excited at 810 nm in the same conditions involved participation from a second, additional cofactor, that we identify with BM. The present, selective RR data on BL fully confirm an earlier interpretation of difference, Soret resonant Raman spectra (Robert, B.; Lutz, M. Biochemistry 1988, 27, 5108−5114), according to which a H-bond engaged with water by the keto group of BL should significantly strengthen upon formation of the P•+ radical state of the primary donor. The present data also indicate that the equivalent H-bond engaged by the keto group of BM should strengthen as well, however by about half the enthalpy change of the L-side bond. The absolute wavenumbers, and compared P•+-induced shifts of the stretching modes of the acetyl CO groups of BL and BM are interpreted as indicating (i) that the acetyl carbonyl of BL is located in a higher permittivity environment than that of BM and (ii) that the frequency of the acetyl CO vibrator of BM may accordingly experience a stronger electric field effect from the P•+ radical state than that of BL. A set of differences observed between BL and BM spectra specifically concerns their Mg-sensitive modes. These differences are not resulting from a differential field effect, and indicate a sizable difference of conformations between the MgN4Nε2(His) groups of BL and BM. The macroring core sizes of both BL and BM, however, are close to the size of relaxed, five-coordinated BChl a in solution. Finally, comparisons of RR spectra excited at 790, 800, and 810 nm from RCs chemically poised in the P° or P•+ states revealed a sizable contribution from the neutral state of the primary donor in RR spectra excited at 810 nm only. This confirms that in electronic spectra of reduced RCs the 810 nm shoulder should arise in part from the upper excitonic component, Py +, of the primary donor.

Strong Effects of an Individual Water Molecule on the Rate of Light-driven Charge Separation in the Rhodobacter sphaeroides Reaction Center

“…In a similar vein, the effects of the GM203L and GM203D mutations on the absorbance spectrum of the reaction center are consistent both with the expected effects of removal of a hydrogen bond interaction between the keto carbonyl of B A and water A (58 -61) and with the predicted effects of changing the polarity of the environment of the BChl (55,58,62) Nevertheless, irrespective of the mechanism involved, it is clear that water A influences the stretching frequency of the keto carbonyl group of B A . In the oxidized reaction center, the observed 1675 cm Ϫ1 frequency provides unambiguous evidence for the presence of a hydrogen bond between water A and the keto carbonyl of B A (12,26). This is observed both when the reaction center is chemically oxidized to form P ϩ , as in the present work (Fig.…”

“…5. As discussed previously (26), the resonance Raman spectrum of untreated or chemically reduced reaction centers is strongly distorted above 1100 cm Ϫ1 by fluorescence emission from P, but this emission band is lost when P is oxidized to P ϩ by treatment with ferricyanide. As has been described (26), the spectrum obtained with 800 nm excitation of ferricyanide-oxidized reaction centers comprises bands arising from B A .…”

“…In both cases sodium ascorbate and phenazine methosulfate were added to final concentrations of 1 mM and 25 M, respectively, to ensure full reduction of the P BChls. Resonance Raman spectra were recorded at 77 K from samples of purified reaction centers as described recently (26). Femtosecond transient absorbance difference spectra were also recorded using antenna-deficient membranes as described in detail recently (27,28).…”

Resonance Raman spectroscopy

“…In resonance conditions, the large gain in the Raman signal allows easier measurements on biological samples, although it is most often impossible to excite photosynthetic complexes in their red transitions, as their intrinsic fluorescence usually impairs the observation of the Raman signal. This was achieved up to now only in the case of bacterial reaction centers, mainly for observing the spectra of the accessory bacteriochlorophyll (Frolov et al 2002) or the very low frequencies of the primary electron donor (Cherepy et al 1994). On the other hand, since Raman-active molecular vibrations are those involving change in molecular polarizability, the Raman signal of water thus seldom interferes with that of the biological molecules being studied.…”

Low-frequency resonance Raman studies of the H(M202)G cavity mutant of bacterial photosynthetic reaction centers

“…Certain bands in this region are sensitive to the structure of BChl; however, modes in the very low-frequency region (<500 cm )1 ), which include out-of-plane deformations of the macrocycle, substituent deformations, and metal-ligand stretching modes are generally more sensitive to structural changes than the mid-to high-frequency in-plane stretching and bending vibrations of the macrocycle (Palaniappan et al 1995;Laporte et al 1996;Czarnecki et al 1997aCzarnecki et al , b, c, 1999Chen et al 2004Chen et al , 2005. In this connection, we and others have previously examined the low-frequency resonance Raman (RR) spectra of the BChl and BPh cofactors in a variety of wild-type and genetically modified RCs (Cherepy et al 1994(Cherepy et al , 1995(Cherepy et al , 1997aPalaniappan et al 1995;Laporte et al 1996;Czarnecki et al 1997aCzarnecki et al , b, c, 1999Eads et al 2000;Frolov et al 2002;Chen et al 2004Chen et al , 2005. In one series studies, we examined RCs wherein the BChls (P and the accessory pigments) were 15 N or 26 Mg labeled and the His residues were 15 N labeled (Czarnecki et al 1997b, c).…”