The initial solar energy conserving event in photosynthesis is the transfer of an electron between an excited donor and a neighboring acceptor molecule in the reaction center, an intrinsic membrane protein-pigment complex. In this project the PI will continue his studies of the purple nonsulfur bacterium Rhodobacter sphaeroides, investigating the driving force and temperature dependence of the initial electron transfer reactions. The PI has used a form of reaction diffusion theory, applied previously to electron transfer in viscous solvents, to describe the complex kinetics of electron transfer as a function of driving force and temperature. This approach has been remarkably successful, implying that protein conformational changes initiated by light absorption control the observed kinetics instead of a static barrier crossing between two potential surfaces. Several important questions have been raised by this work that need to be answered. First, the nature of the protein motion and the spectroscopic signal at 280 nm that appears to be a probe of this motion are unclear. It is currently hypothesized that this is due to tryptophan residues responding to changes in the protein environment, but this remains to be proven. Second, a more detailed mechanistic exploration of the relationship between protein relaxation and electron transfer is necessary. This new model provides an opportunity to determine the reorganization energy, driving force and coupling for a whole series of mutants as a function of temperature, resulting in a much more complete mechanistic picture of initial photosynthetic electron transfer than has ever been available previously. Finally, this work will be merged with current directed evolution approaches to produce mutants that undergo high yield electron transfer along the normally unused cofactor pathway (the B-side). A very similar set of studies as a function of driving force and temperature will then be performed on these mutants to explore the mechanistic similarities and differences between the two electron transfer pathways.
The PI is involved in expanding interdisciplinary research at both the graduate and undergraduate levels. The PI has seven undergraduates working with him, two directly on this project. In addition, the concepts involved in photosynthetic research are used to enrich his teaching in both physical chemistry and biochemistry. For example, the PI teaches a course on bio-nanotechnology as part of a learning community project at ASU in which sophomores explore the science, policy and sociology of nanotechnology. The PI is also the director of an NSF IGERT program in biomolecular nanotechnology. The photosynthetic reaction center is a premier example of an optoelectronic device at the nanoscale and the concepts from this work are one of the key examples of biomolecular nanotechnology studied by the IGERT students. The PI is also the current director of the ASU BioEnergy Research Initiative, a new initiative growing out of ASU's Photosynthesis Center that seeks to take our growing understanding of photosynthetic processes and utilize them in the development of new energy sources and means of energy transduction. Finally, the PI directs the Center for BioOptical Nanotechnology in the Biodesign Institute. In this role, he directly interfaces with a large number of private, commercial and citizens groups, and these discussions form the basis for a new approach to community-embedded research, in which the needs of society and the process of discovery are integrated in a new hybrid model for interdisciplinary research. This project is jointly supported by Molecular Biophysics in the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences and the Experimental Physical Chemistry Program in the Division of Chemistry in the Mathematical and Physical Sciences Directorate.
Project Outcome: The nearly unity quantum efficiency of solar energy conversion in photosynthesis is achieved by the transfer of an electron from an excited donor through a series of acceptor molecules in the reaction center protein, an intrinsic membrane protein-pigment complex. A key aspect of the biological mechanism of photosynthetic energy conversion is the role of the protein environment that facilitates the electron transfer reactions. What we have come to understand in recent years is that the protein does much more than just hold the chemically active cofactors in the right positions. The fact that the protein can move on a large number of time scales makes it a very special kind of solvent for chemical reactions. To explore these ideas, we have taken advantage of specific features of the photosynthetic reaction center from purple nonsulfur bacteria (its structural simplicity, spectral characteristics, and electron transfer on multiple time scales), and utilized it as a "lab on a molecule" to explore the detailed role of protein structure and dynamics in electron transfer using time-resolved laser spectroscopic techniques coupled with high level molecular dynamics calculations. By investigating the rate of the initial and secondary photosynthetic electron-transfer reactions in wild-type and mutant reaction centers of Rhodobacter sphaeroides (both experimentally and via molecular dynamics simulations), we have shown that protein dynamics modulates the rate of each step. Because proteins can move on many different timescales and in specific ways, they can "break" the usual rules of thermodynamics and kinetics that apply to homogeneous solvents like water. Effectively, the ability of the protein to thermally equilibrate with reaction intermediates changes dramatically with the time scale upon which the reaction occurs. As a result, the key energetic parameters that control chemistry, including the activation energy of a reaction that determines how fast it proceeds, become dependent in a fundamental way on the time scale of the reaction. In the photosynthetic reaction center, this means that electron transfer reactions on different time scales can be optimized essentially independently, even though they use the same physical cofactors of the reaction center. This provides evolution with a dimension for functional optimization beyond the static structure and the dynamics of the groups that are directly involved in the reaction mechanism itself. In the study of the initial electron transfer step (P* --> P+HA-), a form of reaction diffusion theory was used to successfully describe the complex kinetics of electron transfer as a function of driving force and temperature in wild type and 14 mutant reaction centers. This study suggested that protein dynamics was rate limiting in the initial electron transfer process. More recently we have explored the secondary electron transfer (P+HA- --> P+QA-), and the recombination of P+HA- in mutants that alter the protein environment near the electron acceptors (BA and HA). This work also indicated that protein dynamics play a major role in controlling the rate and pathway of the various electron transfer reactions, and therefore the overall efficiency. Electron transfer from HA- to QA, which takes place on the time scale of hundreds of picoseconds, is of particular interest in this regard as it involves large-scale collective protein motion. Impact & Benefits: Proteins mediate nearly all of the chemistry of life. The realization that thermodynamics is fundamentally dependent on protein dynamics provides an experimental basis for quantitative modeling of protein structure/dynamics/function relationships, allowing us to predict how protein structure and dynamics will affect the energetics and kinetics of the reactions it mediates. This has significant implications for functional protein design. Protein dynamics in the reaction center makes the solar energy conversion process robust; the system can operate over a wide range of conditions. Clearly much has been done to use our understanding of protein structure and function to optimize catalysis, understand disease and design drugs. By adding the ability to understand and control dynamics in a structurally defined way, the potential for designing or redesigning protein function can be greatly enhanced. Background & Explanation: This research was made possible through NSF MCB support of fundamental research in experimental aspects of protein-mediated chemistry. Our researches primarily used ASU's ultrafast laser facility, also funded by NSF, which acts like a high-speed motion picture camera that can capture data from these lightning-fast reactions.
