EPR and Endor Studies of the Primary Donor Cation Radical in Native and Genetically Modified Bacterial Reaction Centers

Rautter, J.; Geßner, Ch.; Lendzian, Friedhelm; Lubitz, Wolfgang; Williams, Joann; Murchison, H. A.; Wang, S.; Woodbury, Neal W.; Allen, James P.

doi:10.1007/978-1-4615-3050-3_12

Cited by 16 publications

(11 citation statements)

References 9 publications

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“…This observation qualitatively suggests a more asymmetric charge distribution between PL and PM in the photooxidized state of LH(M 160), compared to Wt, and is consistent with the increase in frequency and the slight decrease in intensity of the highwavenumber band at «2800 cm™1 in this mutant (Figure 2b). This proposal is also in good agreement with ENDOR spectroscopy of P+ on isolated RCs indicating that 84% of the spin density resides on Pl+ in LH(M160), compared to 68% in Wt (Huber et al, 1992;Rautter et al, 1992;Feher, 1992).…”

Section: Discussionsupporting

confidence: 79%

“…For LH(L131), it has been proposed that the spin density is shifted toward the M half of the dimer, with 53% present on Pm+ (Rautter et al, 1992). The lack of a detectable effect on the 2600-cm™1 band in LH(L131) (Figure 2c) can thus be rationalized by assuming that the charge asymmetry is reversed in this mutant with respect to Wt, but is similar in magnitude.…”

Section: Discussionmentioning

confidence: 94%

“…These symmetry-related amino acid residues are near the ring V keto carbonyls of Pl and PM, respectively. The two single-site mutants Leu-M160 to His, LH(M160), and Leu-L131 to His, LH(L131), as well as the corresponding double mutant, LH(M 160)+LH(L 131), have been constructed and characterized with respect to their primary donor oxidation potential, initial electron-transfer rates, charge recombination kinetics, early-time thermodynamics (Williams et al, 1992a,b;Woodbury et al, 1993), and spin density distribution between the two halves Pl and Pm in P+ (Rautter et al, 1992). Here, the putative hydrogen bonding of the 9-keto C=0 of Pl and/or Pm to the protondonating His residue is investigated by FTIR spectroscopy.…”

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confidence: 99%

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Fourier transform infrared study of the primary electron donor in chromatophores of Rhodobacter sphaeroides with reaction centers genetically modified at residues M160 and L131

et al. 1993

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Structural changes in chromatophores of Rhodobacter sphaeroides reaction center mutants associated with the substitution of amino acid residues near the primary electron donor P have been investigated by light-induced FTIR difference spectroscopy. The single-site mutations Leu-L131 to His and Leu-M160 to His and the corresponding double mutation were designed to introduce a proton-donating residue that could form a hydrogen bond with the keto carbonyl of ring V of each bacteriochlorophyll (PL and PM) of the dimer. The presence of large positive bands at approximately 1550, 1480, and 1295 cm-1, as well as at 2600-2800 cm-1 in the light-induced P+QA-/PQA FTIR difference spectra, corresponding to the photooxidation of P and the photoreduction of the primary quinone QA, demonstrates that the BChl dimer state of P+ is preserved in the LH(L131), LH(M160), and LH(M160)+LH(L131) mutants, although frequency shifts and amplitude changes can be observed, notably for LH(M160). Compared to wild type, these changes are thought to reflect a different charge repartition over the two BChls in P+. Large frequency downshifts in the 9-keto C=O stretching region of the P+QA-/PQA FTIR difference spectra of chromatophores are observed in the mutant samples relative to wild type. For the LH(M160) mutant, a large differential signal at 1678/1664 cm-1 is assigned to a shift, upon photooxidation, of the 9-keto C=O of PM hydrogen-bonded to His-M160, while that at 1718/1696 cm-1 corresponds to the free 9-keto C=O of PL.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Discussionsupporting

confidence: 79%

Section: Discussionmentioning

confidence: 94%

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confidence: 99%

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Fourier transform infrared study of the primary electron donor in chromatophores of Rhodobacter sphaeroides with reaction centers genetically modified at residues M160 and L131

et al. 1993

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“…It seems likely that the large changes in the oxidation potential arise from electrostatic interactions of P+ with the histidine. In addition to the changes in the energy level, large changes in the electron distribution in P+ due to the introduction of the histidine residues have been demonstrated by changes in the relative electron spin densities on the two halves of P+ as measured by electron-nuclear double resonance and Fourier-transform resonance Raman spectroscopy (16,20).…”

Section: Resultsmentioning

confidence: 99%

Specific alteration of the oxidation potential of the electron donor in reaction centers from Rhodobacter sphaeroides.

Lin

Murchison

Nagarajan

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

272

342

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The effects of multiple changes in hydrogen bond interactions between the electron donor, a bacteriochlorophyll dimer, and htdine residues in the reaction center from Rhodobacter sphaeroides have been igaed. Sitedirected mutations were deiged to add or remove hydrogen bonds between the 2-acetyl groups of the dimer and istidine residues at the setry-related sites Hls-L168 and Phe-M197, and between the 9-keto groups and Leu-L131 and Leu-M160. The addition of a hydrogen bond was correlated with an increase in the dimer midpoint potential. Measurements on double and triple mutants showed that changes In the midpoint potential due to alterations at the individual sites were additive.Midpoint potentials ranging from 410 to 765 mV, compared with 505 mV for wild type, were achieved by various combinations of mutations. The optical absorption spectra of the reaction centers showed relatively minor changes in the position of the donor absorption band, indicating that the addiion of hydrogen bonds to hide pimril destabilized the oxidized state of the donor and had little effect on the excited state relative to the ground state. Despite the change in energy of the charge-separated states by up to 260 meV, the mutant reaction centers were still capable of electron taner to the primary quinone. The increase in midpoint potential was correlated with an increase in the rate of charge recombination from the primary quin , and a fit of these data using the Marcus equation idicated that the reorgn i energy for this reaction is =400 meV higher than the change in free energy in wild type. The mutants were still capable of photosynthetic growth, although at reduced rates relative to the wild type. These results suggest a role for protein-cofactor interactions-n particular, hididonor interactions-In establishing the redox potentials needed for electron transfer in biological systems.Although the oxidation-reduction midpoint potentials of identical cofactors in redox proteins can vary by several hundred millivolts, the specific interactions of the cofactor with the protein that result in the variation in midpoint potential are not well understood. The primary electron donor in reaction centers from the purple photosynthetic bacterium Rhodobacter sphaeroides is a bacteriochlorophyll (Bchl) dimer designated P (reviewed in refs. 1-3). The two Bchls of the dimer, labeled A and B, overlap in ring I, where they are separated by -3.5 A. The midpoint potential of the primary donor is =500 mV in wild-type reaction centers from Rb. sphaeroides (4-6) and is expected to be a critical parameter for electron transfer reactions that involve the donor, as alteration of the potential will result in a change in the driving force for these reactions.Mutagenesis experiments have shown that hydrogen bonds between histidine residues and the conjugated carbonyls of the Bchls in the dimer can alter the midpoint potential by significant amounts (5, 7-10). For each Bchl there are two groups, the 9-keto group of ring V and the 2-acetyl group of ring I, t...

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“…For the first time it was possible to assign ENDOR lines unambiguously to the individual dimer halves of the primary donor "special pair". This work on the electronic structure of the primary donor formed the basis for a large number of further studies on this species, see for example [133,[190][191][192][193][194][195][196], leading to a deeper understanding of the role of the dimer in biological electron transfer [133,197,198]. Subsequently, similar ENDOR studies were performed on the primary donors, P •+ 700 and P •+ 680 , in the reaction centers of oxygenic photosynthesis, PS I and PS II [199][200][201][202].…”

Section: Solution Endor and Triple Resonancementioning

confidence: 90%

Biomolecular EPR Meets NMR at High Magnetic Fields

et al. 2018

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In this review on advanced biomolecular EPR spectroscopy, which addresses both the EPR and NMR communities, considerable emphasis is put on delineating the complementarity of NMR and EPR regarding the measurement of interactions and dynamics of large molecules embedded in fluid-solution or solid-state environments. Our focus is on the characterization of protein structure, dynamics and interactions, using sophisticated EPR spectroscopy methods. New developments in pulsed microwave and sweepable cryomagnet technology as well as ultrafast electronics for signal data handling and processing have pushed the limits of EPR spectroscopy to new horizons reaching millimeter and sub-millimeter wavelengths and 15 T Zeeman fields. Expanding traditional applications to paramagnetic systems, spin-labeling of biomolecules has become a mainstream multifrequency approach in EPR spectroscopy. In the high-frequency/high-field EPR region, sub-micromolar concentrations of nitroxide spin-labeled molecules are now sufficient to characterize reaction intermediates of complex biomolecular processes. This offers promising analytical applications in biochemistry and molecular biology where sample material is often difficult to prepare in sufficient concentration for NMR characterization. For multifrequency EPR experiments on frozen solutions typical sample volumes are of the order of 250 μL (S-band), 150 μL (X-band), 10 μL (Q-band) and 1 μL (W-band). These are orders of magnitude smaller than the sample volumes required for modern liquid- or solid-state NMR spectroscopy. An important additional advantage of EPR over NMR is the ability to detect and characterize even short-lived paramagnetic reaction intermediates (down to a lifetime of a few ns). Electron–nuclear and electron–electron double-resonance techniques such as electron–nuclear double resonance (ENDOR), ELDOR-detected NMR, PELDOR (DEER) further improve the spectroscopic selectivity for the various magnetic interactions and their evolution in the frequency and time domains. PELDOR techniques applied to frozen-solution samples of doubly spin-labeled proteins allow for molecular distance measurements ranging up to about 100 Å. For disordered frozen-solution samples high-field EPR spectroscopy allows greatly improved orientational selection of the molecules within the laboratory axes reference system by means of the anisotropic electron Zeeman interaction. Single-crystal resolution is approached at the canonical g-tensor orientations—even for molecules with very small g-anisotropies. Unique structural, functional, and dynamic information about molecular systems is thus revealed that can hardly be obtained by other analytical techniques. On the other hand, the limitation to systems with unpaired electrons means that EPR is less widely used than NMR. However, this limitation also means that EPR offers greater specificity, since ordinary chemical solvents and matrices do not give rise to EPR in contrast to NMR spectra. Thus, multifrequency EPR spectroscopy plays an important role in better understanding paramagnetic species such as organic and inorganic radicals, transition metal complexes as found in many catalysts or metalloenzymes, transient species such as light-generated spin-correlated radical pairs and triplets occurring in protein complexes of photosynthetic reaction centers, electron-transfer relays, etc. Special attention is drawn to high-field EPR experiments on photosynthetic reaction centers embedded in specific sugar matrices that enable organisms to survive extreme dryness and heat stress by adopting an anhydrobiotic state. After a more general overview on methods and applications of advanced multifrequency EPR spectroscopy, a few representative examples are reviewed to some detail in two Case Studies: (I) High-field ELDOR-detected NMR (EDNMR) as a general method for electron–nuclear hyperfine spectroscopy of nitroxide radical and transition metal containing systems; (II) High-field ENDOR and EDNMR studies of the Oxygen Evolving Complex (OEC) in Photosystem II, which performs water oxidation in photosynthesis, i.e., the light-driven splitting of water into its elemental constituents, which is one of the most important chemical reactions on Earth.

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EPR and Endor Studies of the Primary Donor Cation Radical in Native and Genetically Modified Bacterial Reaction Centers

Cited by 16 publications

References 9 publications

Fourier transform infrared study of the primary electron donor in chromatophores of Rhodobacter sphaeroides with reaction centers genetically modified at residues M160 and L131

Fourier transform infrared study of the primary electron donor in chromatophores of Rhodobacter sphaeroides with reaction centers genetically modified at residues M160 and L131

Specific alteration of the oxidation potential of the electron donor in reaction centers from Rhodobacter sphaeroides.

Biomolecular EPR Meets NMR at High Magnetic Fields

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