Concordia University

Zazubovits Research Group

Plant photosystem I Top view of plant Photosystem I. Blue: protein. Grey: chlorophylls. Yellow: carotenoids. Red: quinones, Orange: FeS clusters.

The energy needs of the humankind are constantly increasing. Satisfying these needs by burning fossil fuels results in pollution, undesired climate changes and eventual exhaustion of the fuel reserves. Nuclear energy is not problem-free either. Renewable energy is likely to provide the solution for these challenges. The sun offers the most abundant potential source of energy and delivers 3,850,000x1018 J per year to Earth, compared to ~630 x1018 J per year currently consumed by the human civilization. Sun energy was supporting the life on Earth for billions of years via process of photosynthesis. (In fact, all existing fossil fuel reserves originate from photosynthesis processes of long ago.) As a result of evolution, proteins performing the first steps of photosynthesis achieved amazing effectiveness. Can we learn something useful from Nature by exploring how these first steps are organized? To this end, we explore energy and primary charge separation processes in isolated photosynthetic proteins.

Bacterial reaction center Side view of the bacterial reaction center (BRC). Grey: bacteriochlorophylls. Dark-blue: bacteriopheophytines, Red: quinones, Orange: non-heme iron. Nobel Prize in Chemistry was awarded for determining BRC structure in 1988

Proteins involved in photosynthesis can also serve as model systems in the studies of some very general protein features. Proteins perform wide variety of tasks in the living organisms. Their ability to perform the designated tasks depends critically on their tertiary structure, achieved as a result of folding. But how do proteins “know” how to fold properly? It is well known that they fold to minimize their free energy (including electrostatic and entropic terms). However, folding even a small protein by trial and error would take forever (Levinthal paradox). Thus, proteins are believed to possess funnel-like energy landscapes with multiple hierarchal tiers. These landscapes can be explored using optical spectroscopy. Proteins involved in photosynthesis have in-built fluorescent molecules (chlorophylls) that can serve as very sensitive probes to local protein environment. Optical spectroscopy also allows one to explore heat transport in proteins and through protein-water interfaces.

Photosynthetic reaction centers utilize light energy to transfer electrons through the (thylakoid or cell) membrane. A number of substances can inhibit this process. (The strongest of these substances are agricultural herbicides.) We have demonstrated inhibition of electron transfer in isolated photosynthetic reaction centers by explosives and are continuing to work on photosynthesis-based explosives biosensors.

Single photosynthetic complex spectroscopy and spectral hole burning Relationship between single photosynthetic complex spectroscopy and spectral hole burning. The raster-scan image of the sample containing multiple complexes (of the same type, e.g. LH2) is in the center. One can select a single complex (left, one complex contains multiple chlorophyll molecules, hence multiple spectral lines, © Köhler’s Group, Bayreuth), or one can work with the whole ensemble (right) and select molecules in resonance with the narrow-band laser. Then a small conformational change of the environment may be triggered by light, shifting the absorption of these molecules.

We are capable of spectroscopic measurements on both single molecules and macroscopic samples. Mostly we employ so-called site-selective sub-ensemble spectroscopy techniques (non-photochemical spectral hole burning - in protein or amorphous solids context this involves small conformational changes triggered by light; fluorescence line narrowing, etc). We are also developing various software tools to model single-molecule and site-selective spectroscopy results. Currently we are working on bridging the size-scale gap and engaging in the studies of sub-micron biological samples utilizing Convex-Lens Induced Confinement technique (credit for inspiration to Sabrina Leslie of McGill). The distinguishing feature of our approach is taking CLIC to cryogenic temperatures. On the other hand, at room temperature CLIC cell can be used as tunable-size micro- or nano-fluidic cell for biosensor applications.

Couple of years ago we started expanding our research to graphene and carbon nanotubes (in collaboration with Alexandre Champagne’s group). These systems exhibit a number of interesting optical/spectroscopic properties, particularly in quantum dot regime. Of special interest are simultaneous optical and electron transport measurements.

  • High-resolution tunable dye laser system; 650-730 nm (Sirah Matisse pumped by Spectra-Physics Millennia 6W 532 nm green laser)
  • Tunable Titanium-Sapphire laser (Spectra-Physics; 720-1000 nm, pumped by green laser)
  • Helium bath optical cryostat (1.9 K-300 K, Ukrainian Academy of Sciences)
  • Closed-cycle optical cryostat (8K-300K, Cryomech, with optical cell for biological samples)
  • Single-molecule / single nanoparticle imaging, detection and spectroscopy capability (fluorescence and Raman). Princeton Instruments imaging spectrograph and CCD, Perkin-Elmer APD, home-built microscope where microscope objectives can be placed together with the sample inside the cryostat.
  • The above setup can be converted in literally minutes (by moving pre-aligned modules) to allow for a wide range of high-resolution experiments on macroscopic samples: fluorescence, fluorescence excitation, transmission, etc.
  • Capability to manipulate polarization of light (Meadowlark).
  • Access to Cary 5000 UV-VIS-NIR spectrometer (located in SP 365.11).
  • What if the above Cary is busy or its design limits the desired experiment geometry too much? Probing beam is too strong and causes charge separation in photosynthetic reaction centers? No problem. We developed a home-built tunable light source based on a Horiba 640 spectrograph, featuring resolution of 0.04 nm, better than that of a Cary, and a fraction of a nanowatt/cm2 probing beam.
  • Electrochemical Workstation (CH Instruments).
  • In construction: Convex Lens-Induced Confinement (CLIC) spectroscopy / microscopy, including at cryogenic temperatures.
Representative publications
  1. Levenberg, A. et al. Probing Energy Landscapes of Cytochrome b 6 f with Spectral Hole Burning: Effects of Deuterated Solvent and Detergent. J. Phys. Chem. B 121, 9848–9858 (2017). doi:10.1021/acs.jpcb.7b07686
  2. Herascu, N. et al. Spectral Hole Burning in Cyanobacterial Photosystem I with P700 in Oxidized and Neutral States. J. Phys. Chem. B 120, 10483–10495 (2016). doi:10.1021/acs.jpcb.6b07803
  3. Najafi, M. & Zazubovich, V. Monte Carlo Modeling of Spectral Diffusion Employing Multiwell Protein Energy Landscapes: Application to Pigment–Protein Complexes Involved in Photosynthesis. J. Phys. Chem. B 119, 7911–7921 (2015). doi:10.1021/acs.jpcb.5b02764
  4. Najafi, M., Herascu, N., Shafiei, G., Picorel, R. & Zazubovich, V. Conformational Changes in Pigment–Protein Complexes at Low Temperatures—Spectral Memory and a Possibility of Cooperative Effects. J. Phys. Chem. B 119, 6930–6940 (2015). doi:10.1021/acs.jpcb.5b02845
  5. Zazubovich, V. & Jankowiak, R. Biophotonics of Photosynthesis. in Photonics: scientific foundations, technology and applications (ed. Andrews, D. L.) 129–164 (John Wiley & Sons, Inc., 2015). ISBN 978-1-118-22552-3
  6. Zazubovich, V. Fluorescence Line Narrowing and Δ-FLN Spectra in the Presence of Excitation Energy Transfer between Weakly Coupled Chromophores. J. Phys. Chem. B 118, 13535–13543 (2014). doi:10.1021/jp509056z
  7. Herascu, N. et al. Modeling of Various Optical Spectra in the Presence of Slow Excitation Energy Transfer in Dimers and Trimers with Weak Interpigment Coupling: FMO as an Example. J. Phys. Chem. B 118, 2032–2040 (2014). doi:10.1021/jp410586f
  8. Najafi, M. et al. Spectral Hole Burning, Recovery, and Thermocycling in Chlorophyll–Protein Complexes: Distributions of Barriers on the Protein Energy Landscape. J. Phys. Chem. B 116, 11780–11790 (2012). doi:10.1021/jp308055r
  9. Herascu, N. et al. Effects of the Distributions of Energy or Charge Transfer Rates on Spectral Hole Burning in Pigment–Protein Complexes at Low Temperatures. J. Phys. Chem. B 115, 15098–15109 (2011). doi:10.1021/jp208142k
  10. Jankowiak, R., Reppert, M., Zazubovich, V., Pieper, J. & Reinot, T. Site Selective and Single Complex Laser-Based Spectroscopies: A Window on Excited State Electronic Structure, Excitation Energy Transfer, and Electron–Phonon Coupling of Selected Photosynthetic Complexes. Chem. Rev. 111, 4546–4598 (2011). doi:10.1021/cr100234j
  11. Bhalla, V., Zhao, X. & Zazubovich, V. Detection of explosive compounds using Photosystem II-based biosensor. Journal of Electroanalytical Chemistry 657, 84–90 (2011). doi:10.1016/j.jelechem.2011.03.026
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