Neutron Scattering in Photosynthesis Research

 

The basic principles and selected applications of neutron scattering in photosynthesis research are discussed and reviewed in [1,2]. Briefly, neutrons are elementary particles whose de Broglie wavelengths of about 2 to 10 Å fall in the range of interatomic distances, while their energies of about 1 to 20 meV are in the order of low-energy dynamical excitations, like localized diffusive motions of small protein residues, methyl group rotations or low-frequency protein vibrations. Therefore, neutron scattering techniques can be used for investigations of both structural and dynamical aspects. Since neutrons have no charge, they interact directly with the nuclei of a sample and penetrate deeply into matter, thus providing information on the bulk sample in its specific environment under in vivo or close to in vivo conditions. Complementary to X-ray techniques, neutron scattering is especially sensitive to light atoms, like hydrogen, which is almost homogeneously distributed in biomolecules. Neutron scattering cross sections may vary significantly upon isotope exchange, most particularly deuterium for hydrogen, paving the way to contrast variations in order to highlight scattering from specific sample parts or to switch on and off scattering from hydration water. Our main research interest is the use of coherent and incoherent neutron scattering methods to study the molecular dynamics and structure of photosynthetic pigment-protein complexes or membranes [1,2].

Neutron spectroscopy: In this regard, neutron spectroscopy with energy and momentum resolution – also referred to as quasielastic or inelastic neutron scattering (QENS or INS, respectively) - provides insight into dynamical properties of biomolecules by measuring energy quanta exchanged between incident neutron and sample. QENS has already been used to characterize the dynamical properties of purple membrane, PS II membrane fragments and different photosynthetic protein complexes and revealed intriguing correlations between dynamics and biological functions. The major results include:

1.)In the case of photosystem II membrane fragments of green plants, quasielastic neutron scattering (QENS) has established that the onset of molecular motions on the picoseconds timescale at ~240 K [3], and at a relative humidity of ~45% [4] is strictly correlated with the temperature- and hydration-dependent electron transfer efficiency from an electron donor referred to as QA to a transiently bound acceptor molecule named QB. Such a characteristic dependence of a functional process on temperature and hydration indicates a crucial role of molecular dynamics in the underlying molecular mechanism [3,4].

2.)Functional activity and molecular dynamics can be simultaneously monitored in photosensitive biomolecular systems using the time-resolved laser-neutron pump-probe technique [5,6]. The principle of a time-resolved QENS experiment is illustrated in Figure 6. The neutron probe pulse (n) tests the sample in a functional state initiated by the laser pulse.

3.)Inelastic neutron scattering can be used for vibrational spectroscopy of photosynthetic antenna complexes up to physiological temperatures and is thus complementary to low-temperature site-selective optical spectroscopy [7,8].

Small-angle neutron scattering: furthermore, small-angle neutron scattering (SANS) is a non-invasive experimental tool providing structural information on biological systems in aqueous environment without a need for staining or fixation. SANS can also be used to investigate structural responses to external or environmental parameters in real time with a time resolution in the range of seconds. SANS has already been used to study the ultrastructure of cyanobacterial, algal and plant thylakoid membranes and their reorganizations during different photosynthetic processes as well as to investigate the hydration dependent lamellar spacing of photosystem II membrane fragments (see Figure 7).

Contribution to ESS: The European Spallation Source (ESS) to be built in Lund, Sweden as a collaborative effort of 17 European countries is a new class of neutron source with unparalleled flux and brightness. Our group has managed to conclude the first in-kind contract with ESS in Europe at all in order to built a setup for time-resolved laser-neutron pump-probe experiments that will be adapted to several different ESS instruments.

 

References:

  1. Nagy, G., Garab, G., and Pieper, J., Neutron Scattering in Photosynthesis Research, in: Contemporary Problems of Photosynthesis (Editors: S. Allakhverdiev, A. B. Rubin, V. A. Shuvalov) Izhevsk Institute of Computer Science, Izhevsk–Moscow, 2014, Vol. 1, p. 69–121.
  2. Pieper, J., Renger, G., Protein dynamics investigated by neutron scattering, invited review, Photosynthesis Research (2009), 102 (2-3), 281
  3. Pieper, J., Buchsteiner, A., Hauß, Th., Baczyński, K., Adamiak, K., Lechner, R. E., Renger, G., Temperature- and hydration-dependent protein dynamics in photosystem II of green plants studied by quasielastic neutron scattering, Biochemistry (2007), 46 (40), 11398
  4. J. Pieper, T. Hauß, A. Buchsteiner, G. Renger, Eur. Biophys. J. 37, 657 (2008)
  5. Pieper, J., Buchsteiner, A., Dencher, N. A., Lechner, R. E., Hauß, Th., Transient protein softening during the working cycle of a molecular machine, Physical Review Letters (2008), 100, 228103
  6. Pieper, J., Time-Resolved QENS Studies of Native Photosystems, Biochim. Biophys. Acta - Proteins & Proteomics (2010), 1804 (1), 83
  7. Pieper, J., Irrgang, K.-D., Renger, G., Lechner, R. E., Density of Vibrational States in the Light-Harvesting Complex II of Green Plants Studied by Inelastic Neutron Scattering, Journal of Physical Chemistry B (2004), 108, 10556
  8. Pieper, J., Trapp, M., Skomorokhov, A., Natkaniec, I., Peters, J., Renger, G. Temperature-dependent vibrational and conformational dynamics of photosystem II membrane fragments from spinach investigated by elastic and inelastic neutron scattering Biochim. Biophys. Acta – Bioenergetics (2012), 1817, 1213

Figures:

 

Figure 1. Structure of PSII of Synechococcus elongatus according to Zouni et al. Nature 409, 739 (2001) viewed along the membrane normal. The transmembrane α-helices of the central D1/D2 heterodimer are shown as yellow and orange cylinders, respectively. The core antenna complexes CP43 and CP47 are indicated in purple and red, respectively. The PsbO protein is shown as a green β-sheet structure and Cytochrome  c-550 as a helical model. The cofactor arrangement is shown in more detail in Figure 2. This figure is reprinted from Zouni et al. with permission.

 

 

Figure 2. Arrangement of the cofactors within the PS II RC seen along the membrane normal. Chlorophylls (Chl) are coloured in green, pheophytins (Pheo) in yellow, plastoquinones in light blue and carotenoids in brown, respectively. The red arrows indicate the directions of light-induced charge separation, water splitting, and plastoquinol formation. This figure is reprinted from Müh, et al., Biophys. Acta 1817, 44 (2012) with permission.

 

 

Figure 3. QENS spectra (black diamonds) of PS II membrane fragments hydrated with D2O at 44, 66, and 90% r.h., respectively, obtained at the time-of-flight spectrometer NEAT with an incident neutron wavelength of 5.1 ˚A and an elastic resolution of 93 µeV (about 20 ps) at a scattering angle of 75.3° at 300 K according to [3]. Black full lines show normalized Lorentzian fits. The elastic peak is cut off at a value of 0.5 for ease of inspection. The inset shows a schematic view of the hydrated PS II membrane sample with a) reaction center, b) core and minor antennae and c) major antenna complexes. The hydration water is indicated in purple. This figure is reprinted from [3] with permission.

 

 

Figure 4. Temperature dependence of the slow (QISF1, squares) and fast (QISF2, triangles) quasielastic components obtained from QENS data of PS II membrane fragments hydrated at 90 % r.h. according to [3]. The electron transfer efficiency is shown by a full red line This figure is reprinted from [3] with permission. Copyright (2007) American Chemical Society.

 

 

Figure 5. Conformational changes in the vicinity of the QB binding site upon QAàQB electron transfer according to Mulkidjanian et al. Biochem. Soc. Trans. 33, 845 (2005), reprinted with permission.

 

Figure 6. Schematic representation of time-resolved (laser-neutron) pump-probe experiments: the synchronization of a pulsed QENS measurement (panel I) with laser activation of protein function (panel II). Note that for better illustration the timescale in panel I is linear, but logarithmic in panel II. The scales coincide when the neutron probe pulse n arrives at the sample (see vertical dashed line). Panel I: Scheme of pulsed QENS in a time-of-flight diagram. Monochromatic neutron pulses (see full arrows) are selected by a set of rotating choppers A and B with a repetition time τ. The neutron probe pulse (n) arrives at the sample during the functional state prepared by the laser pulse, while neutron pulses (n - 1) and (n + 1) arrive before and after the laser-induced state of the sample, respectively. The dashed arrows indicate the neutron flight paths after interaction with the sample. Panel II: Flash-induced fluorescence quantum yield of an intact spinach leaf (red line) and of PSII membrane fragments with inhibited QA → QB electron transfer hydrated at 90% r.h. (blue line). The actinic laser flashes at t=0 had a wavelength of 532 nm and a pulse energy of 8 mJ/cm2. The decay of this curve is mainly determined by QA→QB electron transfer and, thus, defines the timescale of the PS II laser-QENS experiment. Black diamonds indicate the arrival times of the neutron pulses with a repetition time τ at the sample position. The value Δt denotes the time delay between laser flash and the neutron pulse. This figure is reprinted from [8] with permission.

 

Figure 7. Repeat distance D of PS II membrane fragments derived from the diffraction data as a function of hydration level (red points) and a representative error bar. The inset shows a schematic representation of PSII membrane fragments with a) reaction center, b) core and minor antenna complexes and c) major antenna complex LHC II. Membrane extrinsic parts at the luminal side of PS II are represented by white ovals, while hydration water is depicted in purple (bound water) and light blue (bulk water). Note that the membrane fragments shown are associated on their stromal side.