ENDOR Studies of the Primary Donor Cation Radical in Mutant Reaction Centers of Rhodobacter sphaeroides with Altered Hydrogen-Bond Interactions

Abstract

The electronic structure of the cation radical of the primary electron donor was investigated in genetically modified reaction centers of Rhodobacter sphaeroides. The site-directed mutations were designed to add or remove hydrogen bonds between the conjugated carbonyl groups of the primary donor, a bacteriochlorophyll dimer, and histidine residues of the protein and were introduced at the symmetry-related sites L168 His-->Phe, HF(L168), and M197 Phe-->His, FH(M197), near the 2-acetyl groups of the dimer and at sites M160 Leu-->His, LH(M160), and L131 Leu-->His, LH(L131), in the vicinity of the 9-keto carbonyls of the dimer. The single mutants and a complete set of double mutants were studied using EPR, ENDOR, and TRIPLE resonance spectroscopy. The changes in the hydrogen bond situation of the primary donor were accompanied by changes in the dimer oxidation midpoint potential, ranging from 410 to 710 mV in the investigated mutants [Lin, X., Murchison, H. A., Nagarajan, V., Parson, W. W., Williams, J. C. & Allen, J. P. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10265-10269]. It was found that the addition or removal of a hydrogen bond causes large shifts of the spin density between the two halves of the dimer. Measurements on double mutants showed that the unpaired electron can be gradually shifted from a localization on the L-half of the dimer to a localization on the M-half, depending on the hydrogen bond situation. As a control, the effects of the different hydrogen bonds on P.+ in the mutant HL(M202), which contains a BChlL-BPheM heterodimer as the primary donor with localized spin on the BChl aL [Bylina, E. J., & Youvan, D. C. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7226-7230; Schenck, C. C., Gaul, D., Steffen M., Boxer S. G., McDowell L., Kirmaier C., & Holten D. (1990) in Reaction Centers of Photosynthetic Bacteria (Michel-Beyerle M. E., Ed.) pp 229-238, Springer, Berlin] were studied. In this mutant only small local changes of the spin densities (< or = 10%) in the vicinity of the hydrogen bonds were observed. The effects of the introduced hydrogen bonds on the spin density distribution of the dimer in the mutants are discussed in terms of different orbital energies of the two BChl a moieties which are directly influenced by hydrogen bond formation. The observed changes of the spin density distribution for the double mutants are additive with respect to the single mutations.(ABSTRACT TRUNCATED AT 400 WORDS)

EPR, ENDOR, and Special TRIPLE measurements of P•+ in wild type and modified reaction centers from Rb. sphaeroides

“…Since the hfcs of the b-protons at positions 7, 8, 17, and 18 (Fig. 1c) are strongly dependent on the geometry of the respective hydrated rings (Rautter et al 1995), the EPR linewidth may be changed even without a spin redistribution between the two halves of the dimer. More definitive conclusions can, therefore, only be drawn if the resolution is increased significantly, e.g., by double and triple resonance experiments, yielding the individual nuclear hyperfine coupling constants.…”

“…Mutants with the number of hydrogen bonds to the conjugated system of P ranging from zero to four have midpoint potentials from 410 to 765 mV, compared to 505 mV for wild type (Lin et al 1994). These mutants also show significant shifts in the spin density distribution over the two halves of P (Rautter et al 1995;Artz et al 1997;Müh et al 2002). The shifts of the P/P •?…”

“…by mapping the spin density distribution over the conjugated system. In wild type, the distribution is asymmetric with more of the spin density being located on the L-side of P (P L ) than the M-side (P M ) (Geßner et al 1992;Lendzian et al 1993;Rautter et al 1994;1995;Artz et al 1997;Müh et al 2002;Lubitz et al 2002). Due to the large number of protons in the BChl macrocycle (Fig.…”

“…(c) Molecular structure of bacteriochlorophyll a (BChl a) with IUPAC numbering; the two methyl groups 2 1 and 12 1 and the b-protons 7, 8, 17, and 18 are indicated studies of P •? in RCs both in liquid solution and single crystals, several hfcs have been resolved and unambiguously assigned (Geßner et al 1992;Lendzian et al 1993;Artz et al 1997;Rautter et al 1994;1995;Müh et al 2002). In general, for samples in liquid solutions, the technique of Special TRIPLE is well suited to obtain highquality spectra that can be used to gain detailed insight into the spin and charge distribution within P •?…”

“…. These techniques have also been used to investigate the effect of a number of different mutations in bacterial photosynthetic RCs (Artz et al 1997;Rautter et al 1995;Müh et al 1998;Lubitz et al 2002).…”