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)
The primary electron donor in bacterial reaction centers is a dimer of bacteriochlorophyll a molecules, labeled L or M based on their proximity to the symmetryrelated protein subunits. The electronic structure of the bacteriochlorophyll dimer was probed by introducing small systematic variations in the bacteriochlorophyll-protein interactions by a series of site-directed mutations that replaced residue Leu M160 with histidine, tyrosine, glutamic acid, glutamine, aspartic acid, asparagine, lysine, and serine. The midpoint potentials for oxidation of the dimer in the mutants showed an almost continuous increase up to Ϸ60 mV compared with wild type. The spin density distribution of the unpaired electron in the cation radical state of the dimer was determined by electron-nuclear-nuclear triple resonance spectroscopy in solution. The ratio of the spin density on the L side of the dimer to the M side varied from Ϸ2:1 to Ϸ5:1 in the mutants compared with Ϸ2:1 for wild type. The correlation between the midpoint potential and spin density distribution was described using a simple molecular orbital model, in which the major effect of the mutations is assumed to be a change in the energy of the M half of the dimer, providing estimates for the coupling and energy levels of the orbitals in the dimer. These results demonstrate that the midpoint potential can be fine-tuned by electrostatic interactions with amino acids near the dimer and show that the properties of the electronic structure of a donor or acceptor in a protein complex can be directly related to functional properties such as the oxidation-reduction midpoint potential.The reaction center is the site of the primary process in photosynthesis, which is the conversion of light energy into a charge-separated state (for reviews, see ref. 1). The electron transfer process in the reaction center has the remarkable aspect that the quantum yield is near unity, that is, for every photon absorbed, one electron is transferred. The reaction center isolated from the purple bacterium Rhodobacter sphaeroides is particularly useful for the study of electron transfer because this complex has been well characterized biochemically and spectroscopically, and the three-dimensional structure has been determined by x-ray diffraction (2-5). The reaction center has two core subunits, L and M, that form the binding sites for the cofactors and are related by an Ϸ2-fold symmetry axis. The primary electron donor (P) consists of two symmetry-related bacteriochlorophyll (BChl) a molecules labeled P L and P M that overlap at the ring I position and are separated by Ϸ3.5 Å (Fig. 1). Light absorption causes the excitation of P, and within 3.5 ps, an electron is transferred to a bacteriopheophytin acceptor. Electron transfer proceeds to the primary quinone acceptor, Q A , in Ϸ200 ps and to the secondary quinone, Q B , in Ϸ200 s. After reduction of the donor by cytochrome c 2 , absorption of a second photon leads to the transfer of a second electron to Q B , which then leaves the protein carry...
The influence of specific protein-cofactor interactions on the electronic structure of the primary donor cation radical Pf ' and the acceptor anion radicals Q;* and Q;' in wild type and mutant reaction centers of photosynthetic bacteria is investigated by ENDOR and Pulsed EPR techniques. The results show that hydrogen bonds to the primary donor have a strong effect on the distribution of the unpaired electron over the two BChl halves of the special pair, P". A correlation between the rate of reduction of P f ' by cytochrome c2 and the spin density distribution within the dimer is found. ESEEM made it possible to detect I4N nuclear quadrupole resonances for QA' and Q;* that are assigned to nitrogen atoms of ligating amino acids (histidines and peptide backbone). The deduced hyperfine couplings indicate significant delocalization of spin density onto these residues. This indicates an active role of these ligands in the electron transfer from QA to QB.
Reaction centers (RCs) from four species of purple bacteria, Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodospirillum rubrum, and the recently discovered bacterium Rhodospirillum centenum, have been characterized by optical spectroscopy [Wang, S., Lin, X., Woodbury, N. W., & Allen, J. P. (1994) Photosynth. Res. (submitted for publication)] and magnetic resonance spectroscopy. All RCs contain a bacteriochlorophyll (BChl) a dimer as the primary donor. For Rb. sphaeroides and Rs. rubrum the donor QY optical band is at approximately 865 nm, compared to approximately 850 nm for Rb. capsulatus and Rs. centenum. The primary donor in the RCs can be converted between these two forms by the addition or removal of charged detergents. The electronic structure of the cation radical of the primary electron donor P+. was investigated in these species using electron paramagnetic resonance (EPR), electron nuclear double resonance (ENDOR), and electron nuclear triple resonance (TRIPLE) spectroscopy. The EPR line widths of P+. vary significantly and the ENDOR and Special TRIPLE spectra reveal drastic differences in the spin density distribution of the dimer for the different species. Reaction centers from Rb. sphaeroides and Rs. rubrum have a slightly asymmetric spin density distribution over the two halves of the dimer. The respective ratios are 2:1 and 1.6:1 in favor of the L-half of the BChl a dimer. In contrast, the spectra of P+. in reaction centers from Rb. capsulatus and Rs. centenum show an almost complete localization of the unpaired electron on the L-half of the dimer (ratio approximately 5:1).(ABSTRACT TRUNCATED AT 250 WORDS)
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