Arnold J. Hoff

Biophysical techniques in photosynthesis

Preface. Part One: Optical Methods. 1. Developments in Classical Optical Spectroscopy J. Amesz. 2. Linear and Circular Dichroism G. Garab. 3. Fluorescence K. Sauer, M. Debreczeny. 4. Ultrafast Spectroscopy of Photosynthetic Systems R. Jimenez, G.R. Fleming. 5. Data Analysis of Time-Resolved Measurements A.R. Holzwarth. 6. Photosynthetic Thermoluminescence as a Simple Probe of Photosystem II Electron Transport Y. Inoue. 7. Accumulated Photon Echo Measurements of Excited State Dynamics in Pigment-Protein Complexes T.J. Aartsma, R.J.W. Louwe, P. Schellenberg. 8. Spectral Hole Burning: Methods and Applications to Photosynthesis N. Raja, S. Reddy, G.J. Small. 9. Infrared and Fourier-Transform Infrared Spectroscopy W. Mantele. 10. Resonance Raman Studies in Photosynthesis - Chlorophyll and Carotenoid Molecules B. Robert. 11. Stark Spectroscopy of Photosynthetic Systems S.G. Boxer. 12. The Photoacoustic Method in Photosynthesis - Monitoring and Analysis of Phenomena which Lead to Pressure Changes Following Light Excitation S. Malkin. Part Two: Magnetic Resonance. 13. Magnetic Resonance: an Introduction A.J. Hoff. 14. Time-Resolved Electron Paramagnetic Resonance Spectroscopy - Principles and Applications H. Levanon. 15. Electron Spin Echo Methods in Photosynthesis Research R.D. Britt. 16. ENDOR Spectroscopy W. Lubitz, F. Lendzian. 17. Optically Detected Magnetic Resonance (ODMR) of Triplet States in Photosynthesis A.J. Hoff. 18. MagicAngle Spinning Nuclear Magnetic Resonance of Photosynthetic Components H.J.M. de Groot. Part Three: Structure and Oxygen. 19. Structure Determination of Proteins by X-Ray Diffraction M. Schiffer. 20. Electron Microscopy E.J. Boekema, M. Rogner. 21. X-Ray Absorption Spectroscopy:Determination of Transition Metal Site Structures in Photosynthesis V.K. Yachandra, M.P. Klein. 22. Mossbauer Spectroscopy P.G. Debrunner. 23. Characterization of Photosynthetic Supramolecular Assemblies Using Small Angle Neutron Scattering D.M. Tiede, P. Thiyagarajan. 24. Measurements of Photosynthetic Oxygen Evolution H.J. van Gorkom, P. Gast.

On the magnetic field dependence of the yield of the triplet state in reaction centers of photosynthetic bacteria

The yield of the triplet state in reaction centers of Rhodopseudomonas sphaeroides is dependent on the strength of an applied magnetic field. Reaction centers of the wild type that lack a functional iron complexed to the primary acceptor ubiquinone show a dependence similar to that of reaction centers of the mutant R-26 in which the iron-ubiquinone complex is intact. Apparently, the iron of the iron-ubiquinone complex is not essential to the effect, but it does exert an influence on its extent. Inchromatophores, the effect is about 2-fold decreased; the value of the magnetic field at which half the effect is found is about 500 G, in contrast to this value for reaction centers, which is 50--100 G. The magnetodependence of the triplet yield is discussed in terms of the Chemically Induced Dynamic Electron Polarization mechanism (CIDEP).

Electron paramagnetic resonance of spin-correlated radical pairs in photosynthetic reactions

Abstract Previous attempts to interpret the time-resolved electron paramagnetic resonance spectra of photosynthetic bacteria have been based on the premise that electron spin polarization arises in the primary radical pair (P+I− fromed by photoinduced charge separation. The observed spectrum is assumed to be the sum of the EPR spectra of P+ and X−, the radical produced from I− by electron transfer. Here it is argued that P+I− may be too short-lived to give rise to significant polarization and that the experimental spectrum is consistent with the rapid formation of a spin-correlated secondary radical pair.

High-resolution optical absorption-difference spectra of the triplet state of the primary donor in isolated reaction centers of the photosynthetic bacteria Rhodopseudomonas sphaeroides R-26 and Rhodopseudomonas viridis measured with optically detected magnetic resonance at 1.2 K

Abstract We have recorded triplet optical absorption-difference spectra of the reaction center triplet state of isolated reaction centers from Rhodopseudomonas sphaeroides R-26 and Rps. viridis with optical absorption-detected electron spin resonance in zero magnetic field (ADMR) at 1.2 K. This technique is one to two orders of magnitude more sensitive than conventional flash absorption spectroscopy, and consequently allows a much higher spectral resolution. Besides the relatively broad bleachings and appearances found previously (see, e.g., Shuvalov V.A. and Parson W.W. (1981) Biochim. Biophys. Acta 638, 50–59) we have found strong, sharp oscillations in the wavelength regions 790–830 nm ( Rps. sphaeroides ) and 810–890 nm ( Rps. viridis ). For Rps. viridis these features are resolved into two band shifts (a blue shift at about 830 nm and a red shift at about 855 nm) and a strong, narrow absorption band at 838 nm. For Rps. sphaeroides R-26 the features are resolved into a red shift at about 810 nm and a strong absorption band at 807 nm. We conclude that the appearance of the absorption bands at 807 and 838 nm, respectively, is due to monomeric bacteriochlorophyll. Apparently, the exciton interaction between the pigments constituting the primary donor is much weaker in the triplet state than in the singlet state, and at low temperature the triplet is localized on one of the bacteriochlorophylls on an optical time scale. The fact that for Rps. sphaeroides the strong band shift and the monomeric band found at 1.2 K are absent at 293 K and very weak at 77 K indicates that these features are strongly temperature dependent. It seems, therefore, premature to ascribe the temperature dependence between 293 and 77 K of the intensity of the triplet absorption-difference spectrum at 810 nm (solely) to a delocalization of the triplet state on one of the accessory bacteriochlorophyll pigments.

Evidence for a new early acceptor in Photosystem I of plants. An ESR investigation of reaction center triplet yield and of the reduced intermediary acceptors

The yield of the triplet state of the primary electron donor of Photosystem I of photosynthesis (PT-700) and the characteristic parameters (g value, line shape, saturation behavior) of the ESR signal of the photoaccumulated intermediary acceptor A have been measured for two types of Photosystem I subchloroplast particles: Triton particles (TSF 1, about 100 chlorophyll molecules per P-700) that contain the iron-sulfur acceptors FX, FB and FA, and lithium dodecyl sulfate (LDS) particles (about 40 chlorophyll molecules per P-700) that lack these iron-sulfur acceptors. The results are: (i) In Triton particles the yield of PT-700 upon illumination is independent of the redox state of A and of FX,B,A and is maximally about 5% of the active reaction centers at 5 K. The molecular sublevel decay rates are kx = 1100 s−1 ± 10%, ky = 1300 s−1 ± 10% and kz = 83 s−1 ± 20%. In LDS particles the triplet yield decreases linearly with concentration of reduced intermediary acceptors, the maximal yield being about 4% at 5 K assuming full P-700 activity. (ii) In Triton particles the acceptor complex A consists of two acceptors A0 and A1, with A0 preceding A1. In LDS particles at temperatures below −30°C only A0 is photoactive. (iii) The spin-polarized ESR signal found in the time-resolved ESR experiments with Triton particles is attributed to a polarized P-700-A−1 spectrum. The decay kinetics are complex and are influenced by transient nutation effects, even at low microwave power. It is concluded that the lifetime at 5 K of P-700A0A−1 must exceed 5 ms. We conclude that PT-700 originates from charge recombination of P-700A−0, and that in Triton particles A0 and A1 are both photoaccumulated upon cooling at low redox potential in the light. Since the state P-700AF−X does not give rise to triplet formation the 5% triplet yield in Triton particles is probably due to centers with damaged electron transport.

ESEEM study of spin-spin interactions in spin-polarised P+QA− pairs in the photosynthetic purple bacterium Rhodobacter sphaeroides R26

Abstract Electron spin echo envelope modulation (ESEEM) of the transient spin-polarised P+QA− pairs has been studied at 20 and 220 K. The observed strong out-of-phase modulation is interpreted as resulting from electron-electron dipolar and exchange interactions. Fourier-transformed echo envelopes are consistent with the theory developed recently by Tang, Thurnauer and Norris and allow us to obtain readily the values of dipolar and exchange couplings. The results are insensitive to 15N enrichment.

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.

High-resolution triplet-minus-singlet absorbance difference spectrum of photosystem II particles

The triplet state of the primary donor of photosystem II particles prepared from a mutant of Chlamydomonas reinhardtii has been studied at 1.2 K with absorbance‐detected ESR in zero‐magnetic field (ADMR). Two sets of resonances with slightly different zero‐field splitting parameters |D| and |E| were observed, |D| = 285.5, |E| = 38.8 and |D| = 288.8 × 10−4 cm−1, |E| = 42.2 × 10−4 cm−1, respectively. Both sets of |D| and |E| values are close to those found for PT‐700, as are the sublevel decay rates k x = 930 ± 40, k y = 1088 ± 50 and k z = 110 ± 5 s−1. The AMDR‐detected triplet‐minus‐singlet absorbance difference spectrum of PT‐680 is very similar to that of PT‐700 and closely resembles that of covalently connected Chl a dimers in vitro. We conclude that P‐680 is a Chl a dimer whose general structure is similar to that of P‐700.

Carotenoid triplet yields in normal and deuterated Rhodospirillum rubrum

Quantum yields of carotenoid triplet formation in Rhodospirillum rubrum wild type and fully deuterated cells and chromatophores were determined in weak laser flashes for excitation wavelength lambda i = 530 nm (mainly absorbed by the carotenoid spirilloxanthin) and for lambda i = 608 nm (mainly absorbed by bacteriochlorophyll) in the presence and absence of magnetic fields. All experiments were performed at room temperature and in the absence of oxygen. The quantum yield of reaction center bacteriochlorophyll oxidation in wild type preparations, in which all reaction centers are in state PIX, at lambda i = 608 nm is close to unity, whereas the quantum yield of antenna carotenoid triplet formation is low (about 5%); P is the primary electron donor, a bacteriochlorophyll dimer, I the primary acceptor, a bacteriopheophytin, and X the secondary acceptor, an iron-ubiquinone complex. In cells in which the reaction centers are in the state P+IX(-), the antenna carotenoid triplet yield is about 0.2. In contrast, at lambda i = 530 nm, the quantum yield of P+ formation is relatively low (0.3) and the yield of the antenna carotenoid triplet state in state PIX unusually high (0.3). At increasing light intensities of 530 nm only about 3 carotenoids per reaction center of the 15 carotenoids present are efficiently photoconverted into the triplet state, which indicates that there are two different pools of carotenoids, one with a low efficiency for transfer of electronic excitation to bacteriochlorophyll and a high yield for triplet formation, the other with a high transfer efficiency and a low triplet yield. The absorption difference spectrum of the antenna carotenoid triplet, excited by 608 or 530 nm light in the state P+IX(-) does not show the peak at 430 nm, that is present in the difference spectrum of the reaction center carotenoid triplet, mainly observed at lambda i = 608 nm with weak flashes. The yield of the reaction center carotenoid triplet, generated in chromatophores in the state PIX(-), is decreased by about 10% by a magnetic field of 0.6 T. In a magnetic field of 0.6 T the yield of the carotenoid triplet, formed by 530 nm excitation in chromatophores at ambient redox potential, is decreased by about 45%. The high quantum yield of formation and the pronounced magnetic field effect for the carotenoid triplet generated by direct excitation at 530 nm can be explained by assuming that this triplet is not formed by intersystem crossing, but by fission of the singlet excitation into two triplet excitations and subsequent annihilation (triplet pair mechanism), or by charge separation and subsequent recombination (radical pair mechanism). Fully deuterated bacteria give essentially the same triplet yields, both in the reaction center and in the antenna carotenoids and show the same magnetic field effects as non-deuterated samples. This indicates that hyperfine interactions do not play a major role in the dephasing of the spins in the radical pair P+I- nor in the formation of the antenna carotenoid triplet.

Time-resolved ESR and chemically induced dynamic electron polarisation of the primary reaction in a reaction center particle of Rhodopseudomonas sphaeroides wild type at low temperature.

Recently a very fast decaying ESR signal preceding the so-called signal I in plant photosynthesis has been observed [ 1 ] . The signal was emissive, which was taken to provide evidence for the occurrence of chemically induced dynamic electron polarisation (CIDEP). Such spin-polarisation effect may give insight into the events preceding the primary charge separation. Because the primary reactions in the bacterial photosystem are much better understood than those of the plant systems [2], we have investigated light-induced paramagnetism of bacterial reaction centers on a time scale of 20-300 ps. At a temperature of 100°K we have found a fast decaying, partly emissive, flash-induced ESR signal, with a complex timeand field-dependency, in particles obtained from Rhodopseudomonas sphaeroides wild type by treatment with sodium dodecyl sulphate (SDS). We have attempted to analyse the time-resolved spectra in the framework of current CIDEP theories. We can account for the spectral shape by assuming that the radical pair mechanism is operative, through which the ESR signals of the primary donor-acceptor pair become spinpolarised. This polarisation then decays in a time comparable to the spin-lattice relaxation time. Apparently, by monitoring the time evolution of the lightinduced ESR signal one can probe a spin-memory of the much faster events leading to the charge separation.