S. Savikhin: Research Interests

The Photosynthesis

Nature has evolved highly efficient systems for converting sunlight into useful forms of energy for powering the machinery of life. Absorption and conversion of light energy is fundamentally a physical process, and understanding details of photosynthesis cannot be accomplished without applying most advanced physical experimental and theoretical tools. We use steady-state and time resolved spectroscopy tools to visualize the dynamics and path of energy flow and conversion in natural and artificial biomimetic systems, and we apply structure-based model simulation to facilitate the interpretation of the obtained optical data in terms of physical (quantum-mechanical) phenomena that make these processes so efficient.

The photosynthesis has been studied for almost 500 years attracting attention of many distinguished scientists (Robert Hooke, Joseph Priestley, Antoine Lavoisier, Julius von Mayer to name just few). Quite amazingly, the monologue about photosynthesis was even featured in the famous 1938 movie “You can’t take it with you” directed by Frank Capra (with James Stewart starring). While these lines were not part of the original script, it is believed that Frank Capra, who received bachelor’s degree in chemical engineering from Caltech in 1918, included these lines to express his fascination with photosynthesis.

James Stewart on photosynthesis in 1938 movie You Can’t Take it with You.

The recent progress in photosynthesis research owes largely to overall progress in science and technology which made it possible to obtain molecular structures of protein-pigment complexes involved in photosynthesis and to modify these structures using site directed mutagenesis. The function of these molecular machines is governed by quantum mechanics - the traditional area of physical sciences.

The photosynthetic membrane

Photosynthetic process is powered by several photosynthetic proteins that are located within a membrane. The two photosystems (PS I and PS II) convert energy of absorbed photons into a flow of electrons. Both proteins contain significant amount of chlorophylls that absorb sunlight and transfer electronic excitation energy to respective reaction centers that are located in the middle of each complex and that consists of few chlorophyll molecules. The electron generated in PS II complex is transferred to the cytochrome b₆f complex via plastoquinol pool (PQH₂), and then to PS I by a small protein called plastocyanin (PC). The cytochrome b₆f complex couples electron transport to proton translocation across the membrane that serves to build a proton gradient across the membrane. PS II captures its missing electron from water molecule generating free oxygen and protons, which further rises the proton gradient across the photosynthetic membrane. The electrochemical gradient generated in this way drives these protons through an ATP synthase making this molecular size motor spin. The mechanical energy of that motion is used to force ADP and P_i molecules together creating ATP (adenosine triphosphate) that serves as a universal energy carrier in living organisms. The electron generated by PS I is picked up by a small mobile protein – ferredoxin, FD – and is transported to FNR where its energy is used to reduce NADP⁺ into NADPH.

The Photosystem I project.

Photosystem I is one of the main components of the photosynthetic apparatus shared by most photosynthetic organisms. It is located within the photosynthetic membrane and contains about 100 chlorophyll (Chl) molecules. Most of these Chls serve as light harvesting antennae – light absorbed by any of these Chls is stored as electronic excitation of a molecule. The close spacing of the antenna Chls ensures that excitation can be transferred efficiently from Chl to Chl via the resonant Förster energy transfer (FRET) mechanism to the reaction center (RC). The RC is comprised of a set of few closely spaced Chls such that when the RC receives excitation energy, it initiates charge separation and subsequent flow of electrons, which are later picked up by a small mobile protein and the energy is used to reduce an NADPH+ molecule. The quantum efficiency of this light-to-current converter is nearly 100%, which makes it vastly more efficient than any solar cell currently used by humankind. Learning the mechanisms by which nature achieved such a high efficiency will facilitate engineering of biomimetic devices for energy conversion and light detection. In recent years, through experiments and structure-based computer modeling of energy transfer processes, we have established that surprisingly, the overall energetic organization of the antenna pigments is not optimized for the fastest possible energy transfer. This finding relaxes the requirements for an efficient light-converting biomimetic device. Using ultrafast spectroscopy in combination with site-specific mutagenesis, we were also able to reveal which branch of the two almost equivalent branches of the reaction center is active in charge separation and electron transfer (Biophysical Journal 2005). In addition, we measured experimentally the local dielectric constant within the PS I protein (Biophys. J. 2004). This is one of only two experimental publications on the measurement of the dielectric constant within a protein. This effect is essential in understanding the charge transfer process within the protein. Work is under way to understand the role of various protein subunits in PS I. The current state of PS I research is reviewed in chapter 12 of the book “The light-driven plastocyanin, edited by J.H.Golbeck).

In spite of tremendous interest towards the function of PS I and numerous publications, the question of the primary electron donor and of the sequence of the first electron transfer steps is still under intense debate. It has been generally assumed that the special pair of pigments (P700) is the primary donor of the electron, and that within ~1-3 ps upon receiving electronic excitation by P700 the electron is transferred to the “primary electron acceptor” A₀. However, A₀ is too far from P700 for such a rapid electron transfer, and it has been suggested that accessory pigment may, in fact, be a primary electron acceptor, or that the initial electron transfer may initiate from the accessory pigment, and the electron is later donated to it by P700. Spectral congestion (the presence of ~100 antenna Chl a that absorb in the same spectra range) and the fact that the first charge separation step is faster than energy transfer from antenna to RC preclude experimental observation of the electron on the accessory pigment. To tackle this intriguing question we are now engaged it a new project that will use a similar RC from another organism – green sulfur bacterium, where RC is constituted from BChl and Chl pigments whose absorption spectra are drastically different which would allow to visualize the details of the electron transfer process. Even though the structure of that complex has not been determined yet, the protein sequence is similar to that of the protein that houses the PS I RC and thus it is expected that these systems may function in a very similar manner. We are currently collaborating with John Golbeck (Penn State) on that project. We expect that these experiments will reveal the sequence of events in type I reaction center.

PS I movies created by our group:

3D movies of PSI structure, large (80 MB), medium (50 MB), small (14 MB)

Structure based animated simulation of energy transfer in PS I

The Cytochrome b6f project

The cytochrome b6f complex facilitates electron transfer from Photosystem II to PS I and couples it with proton translocation across the membrane. Its function and structure are similar to that of cytochrome bc1, which is a part of the mitochondrial power plant of animals (and humans). The cyt b6f complex was found to contain a single chlorophyll molecule whose function is not yet known. It does not perform any of the usual chlorophyll functions such as light-harvesting and electron transfer. Our group, in collaboration with Prof. W.A. Cramer, is currently focusing on the function of the chlorophyll. It is also known that monomeric chlorophyll is extremely fragile under normal conditions and degrades within a few minutes under sunlight due to the generation of singlet oxygen. It is well known that in photosynthetic proteins,

protection against this is realized by carotenoids placed within ~4 Å of the Chl molecule. This allows efficient triplet energy transfer from Chl to the carotenoid and prevents energy transfer to oxygen with the formation of singlet oxygen.

Our studies revealed that this protection mechanism is not realized in the cyt b6f complex. We proposed two novel mechanisms for this protection (1 and 2). Understanding this protection of Chl and Chl-like pigments is essential for designing durable biomimetic devices.

In other experiments, we were able to estimate the influence of crystal packing on the structure of the protein (in press). This is of general importance since most of the structures available for proteins have been deduced by xray crystallography in a crystalline, not native, environment. We are currently using a supercomputer to perform large scale molecular dynamic simulation of the cyt b6f complex in order to explain the observed phenomena (details of simulation). We also investigate the involvement of oxygen in energy transfer using low temperature experiments (low oxygen mobility is expected to prevent energy transfer) and anoxygenic conditions.

Similar photoprotection mechanisms could be utilized in other areas of science and technology. For example, single molecule fluorescence techniques suffer from chemical photobleaching of a single fluorophore marker. The mechanism of photobleaching is similar to the mechanism of photodegradation of chlorophyll molecules, and it is expected that by applying the same methods that nature used to protect Chl molecules will benefit single molecule techniques. An understanding of the Chl a protection mechanism will facilitate the next and more important question of the function of Chl a in b6f.

The Chlorosome project

We explore the photosynthetic properties of chlorosomes – the largest known antenna systems that contain up to 200,000 chlorophylls and, due to their self-assembling property, are a natural model for artificial light-sensitive devices. Results of recent experiments are summarized in our paper; these include the first observation of triplet excitons in biological systems.

Proposed structure of chlorosome Frigaard, N. et al. 2003 Photosyn. Res. 78: 93-117	A) Chlorobium tepidum cell B) Extracted chlorosomes Frigaard and Bryant, 2004

A chlorosome incorporates its ~200,000 bacteriochlorophyll (BChl) molecules in a single crystal-like structure. It allows photosynthetic organisms to survive in extremely low light conditions, when a single BChl can absorb only one photon every 8 hours. (For comparison, under full sunlight each Chl may absorb up to 10 photons per second). In spite of its enormous size, the quantum efficiency of this light-harvesting antenna is nearly 100%. This system is of special interest since very similar structures could be assembled spontaneously in the laboratory and thus may serve as a part of an efficient biomimetic device. Because of its large size, the detailed structure of this complex is unknown. We are currently collaborating with Prof. D. Bryant (Penn State) in these studies.

Our latest experiments revealed that carotenoids in this complex do not play a crucial photoprotective role, as had been suggested. The results on native carotenoid-lacking mutants of chlorosomes as well as on artificial BChl complexes suggest that strong excitonic triplet-triplet coupling between the closely spaced BChls lowers the triplet excited state energy, resulting in the energy of these triplet excitons being below that of singlet oxygen and thus preventing energy transfer and singlet oxygen formation. Such triplet excitons have not previously been identified in biological systems.

The unique role of triplet excitons in chlorosomes as a novel protection mechanism for biological systems is the focus of our current research.

Oxygen contents in photosynthetic membranes and evolution of oxygenic photosynthesis

Photosynthetic membranes constantly produce oxygen, which implies that oxygen concentration in these membranes may be rather high. Due to high reactivity of oxygen many vital processes in cells depend on oxygen content. However, most of the experiments on proteins extracted from photosynthetic cells are performed either in the presence of ambient oxygen, or in anoxygenic conditions, as the native concentration of oxygen in functioning cells is not known. Since the function of many proteins depends/relies on oxygen, knowledge of proper oxygen concentration is essential for understanding their function. We use diffusion theory and computer modeling to estimate concentration of oxygen in fully functioning photosynthetic organisms, and we explore the ways to measure this quantity within the membranes experimentally. These data should also shed light on evolution of oxygenic photosynthesis. The same approach can be used to tackle a “reverse” problem – oxygen concentration in mitochondria, cellular power plants, which, instead of producing oxygen, consume it.

The Pathogen Detection project

A molecular beacon DNA microarray system for rapid detection of E. coli O157:H7 has been developed in our group in collaboration with Professor Bruce Applegate (Food Sciences, Purdue) and Professor Michael Kane (Department of Computer and Information Technology, Purdue). This research was initiated and supported by a the U.S. Department of Agriculture (USDA).

The method is based on Förster resonant energy transfer (FRET), similar to the method utilized by nature to harvest light energy.

Molecular DNA beacon coils in the absence of target pathogen DNA and remains uncoiled in the presence of target DNA	The fully automated prototype of pathogen detector uses “barcode” format of beacons and can detect hundreds of pathogens in one scan

In the prototype shown in the figure, two highly fluorescent dye molecules are attached to two sides of a DNA probe that is designed to be complementary to the unique fragment of the target pathogen DNA. In the presence of the target DNA, the probe DNA recombines with it, forming a rigid molecule that separates the two dye molecules. In the absence of target DNA, the probe DNA coils, bringing the two dye molecules close so that FRET can occur. In the prototype, a simple green laser is used to excite one of the dye molecules, while a photodetector registers fluorescence from both of the dye molecules separately. The emission ratio from both of the molecules indicates unambiguously the presence or absence of the target DNA and through that the presence or absence of the pathogen in a sample. This approach has been successfully demonstrated in our recent paper, and work is now being done on increasing the photostability of the DNA-dye structures, using the understanding of photoprotection mechanisms found in natural pigment-protein complexes.

The current design employs a “barcode” design where each line on a plate can detect a different pathogen, and the readout detection device scans across the plate in a manner similar to barcode readers. This allows the detection of hundreds of pathogens simultaneously.

Currently this research is extended to develop of a DNA/mRNA based biosensor for the detection/identification of multiple foodborne pathogens from food matrices in a single assay. This will be accomplished by the continued approach of exploiting the specificity of DNA/mRNA sequences associated with the toxins and virulence factors that are unique for the selected agents, using an inexpensive optical detection platform. The proposed multi-disciplinary research project continues to interface the molecular biology of nucleic acid hybridization with the fundamental principles of fluorescence resonance energy transfer. The specific objectives of the research are:

1. Expand the suite of DNA probes to allow the multiplexed detection of Campylobacter spp in an array format.

2. Adapt previously determined multiplex amplification parameters to include the additional probes.

3. Determine parameters for multiplexed detection of mRNA from the targeted foodborne pathogens for determination of viable cells.

4. Validate the beacon approach in a spatial format utilizing a standard microarray reader.

5. Develop a low cost instrument which can analyze the array and validate performance of the system.

Hydrogen generation

Photochemical hydrogen generation from water is one of the cleanest ways to produce hydrogen for future hydrogen fuel cell technology. In that process, light is absorbed by a catalyst and the acquired energy is used to split water into hydrogen and oxygen. One of the promising materials for this process is nanocrystals of iron oxide. A photon absorbed by such a crystal suspended in water creates an electron-hole pair that initiates water splitting. There are several technical challenges in utilizing this material in hydrogen production, and one of them is the fact that the absorption spectrum of this material maximizes at ~600 nm. Even though the absorption band is broad, a significant fraction of solar energy in blue-green region of spectrum may be lost for solar conversion process. We are investigating the ways to broaden the useful spectral absorption of these crystals by attaching suitable molecules that absorb in blue-green region of spectrum and utilize resonant energy transport their electronic excitation energy to the catalyst. This process mimics the design of natural photosynthetic antenna-reaction center systems and is estimated to boost the overall efficiency of the solar conversion process using that technology to ~30%, which is about 3 times higher than the efficiency of the conventional solar cells used in everyday devices.