All of life is based on a small subset of chemical reactions and processes, among the almost infinite number of possible ones. The selected reactions are catalyzed by biological macromolecules that speed up the reaction rates by very many orders of magnitude. The vast majority of these catalysts are proteins known as enzymes, and many of these have small molecules as cofactors that are intimately involved in the chemistry. In biological energy conversions, oxidation-reduction (redox) free energy is converted into transmembrane proton and electrical gradients. The redox chemistry is performed by diverse organic and metal cofactors that are invested with extraordinary properties by the proteins that bind them. The protein-cofactor interactions that yield these properties are the same as those that operate on a substrate in an enzyme active site. Because of the exceptional spectroscopic attributes of redox and photobiological cofactors, the membrane proteins of respiration, photosynthesis and methanogenesis provide ideal systems for studying the essential and defining quality of biochemistry - catalysis with astonishing specificity. This project focuses on the photosynthetic reaction center (RC), which catalyzes a light-driven electron transfer with almost 100% quantum efficiency. The primary aim is to elucidate the mechanisms whereby the photo-activated electron is delivered to the acceptor and stabilized against wasteful backreaction. In large part, this is due to accompanying charge redistributions within the RC, including internal proton transfers and net uptake from solution. The known structure of the RC, its rich spectroscopy, and the light-activatable nature of its reactions allow the origins of these cofactor properties to be characterized in terms of the dynamics and energetics of the molecular structure. In all projects, the impact of chemical and mutagenic perturbations on energetics and kinetics will be studied. Kinetic and equilibrium consequences will be determined by optical absorption spectroscopy, free energy levels by delayed fluorescence and by potentiometry, and structural implications by FTIR, X-ray diffraction and pulsed EPR spectroscopy. Broader impacts: The broader impacts of this research derive from the very diverse methodologies employed, which provide an exceptional training environment for young scientists, with a substantially multidisciplinary component. This work contributes to the practical understanding of processes with global and human impact, including alternative energy sources, biofuels and global warming. They form a strong basis for teaching, mentoring and motivating students in the classroom, at the high school as well as college level.
For biology, external energy is invariably locked up in chemical forms, or in photons, and must be released initially by oxidation-reduction transformations, e.g., in respiration and photosynthesis (this is largely true for energy sources in human society, too). The main contributors in these transformations are membrane-bound enzymes with cofactors (hemes, metal clusters, quinones, flavins, etc) that have extraordinary properties invested in them by the proteins that bind them. Notably, the well-defined X-ray structures of photosynthetic reaction centers (RCs) have elevated this complex to an unprecedented level as a model for understanding electron transfer (ET) in aperiodic materials, excitation energy transfer in pigment arrays and, more recently, proton transfer (PT). This is ideal for our purposes and we use, primarily, the RC of Rb. sphaeroides. Two of its three membrane-protein subunits (L and M) bind all the active cofactors. These respond to light by performing a charge separation that transfers an electron across the membrane to the primary and secondary quinones, QA and QB, which act as electron acceptors. Two light-activated turnovers result in the reduction of QB to the quinol form (QH2). The two quinones are chemically identical (both are ubiquinone-10 - 2,3-dimethoxy-5-methyl-6-decaisoprenyl-1,4-benzoquinone) but are functionally very distinct, which is essential for successful electron transfer between them. In this project, we focus on the redox characteristics of QA and QB, and on the structural and energetic bases of their differences, using a variety of spectroscopic and structural techniques. We are also investigating the biologically critical influence of the membrane environment on the reaction center, as a general model for membrane protein function. 1) Analogs of ubiquinone were synthesized and used to reveal a critical role of the two methoxy groups in determining ubiquinone's unique ability to act as both QA and QB. We found that the 2-methoxy was required for simultaneous activity as QA and QB, while the 3-methoxy group was not. We suggest that this reflects the need for the protein to be able to manipulate distinct orientations (dihedral angles) of the 2-methoxy group in QA and QB function, to tune the redox potentials to allow forward electron transfer. 2) It is well established from computational studies that the orientations of the methoxy groups of UQ affect its electrochemistry, including midpoint potential. However, although many X-ray structures have been solved for the Rb. sphaeroides RC, the methoxy orientations for QA and QB are still ill-defined. Furthermore, the structures only provide information for the oxidized quinone, and equal knowledge of the semiquinone (one electron-reduced) states is needed to define the redox properties. Therefore, in collaboration with Dr. Sergei Dikanov (Illinois), we initiated an extensive program of study using pulsed EPR methods to define the hydrogen-bonding and atomic environments of the QA and QB semiquinones, in wild type and site-specific mutant reaction centers. This work has included 15N- and 13C-labeled RCs and 1H/2H exchange, and has been augmented by computational analyses in collaboration with Professor Patrick O'Malley (Manchester, U.K.). The pulsed EPR techniques, ESEEM (electron spin echo envelope modulation) and HYSCORE (hyperfine sublevel correlation) spectroscopy, are able to provide high resolution structural information on the hydrogen bonding environments and electronic properties of the QA and QB semiquinones. To address the question of methoxy group orientations, we first established the validity of the methods and determined the atomic environments of the binding sites. First we determined the nitrogen environment of the QB semiquinone using 14N and 15N ESEEM and HYSCORE. Next we performed proton ESEEM and HYSCORE on both semiquinones. The QA study was used to validate the method and a novel analysis against known data for QA, while the QB study provided new information on its hydrogen-bonding environment. Finally, we used computational analyses to show that the hydrogen bond model derived from X-ray structures and other experimental approaches was incorrect. This has significant implications for proton and electron transfer events leading to the full reduction to quinol 3) In addition to the methoxy groups of UQ, the redox potentials of QA and QB are controlled by the protein environment. Site directed mutagenesis had shown that substituting polar residues for Ile-M265 (isoleucine at position 265 of the M subunit) in the QA site caused a dramatic lowering of the redox midpoint potential by 80-130 mV. We have now crystallized four of these mutant RCs, with threonine, serine, asparagine or glutamine in place of Ile-M265, and have solved their structures by X-ray crystallography, all to resolutions ≈ 2.8 Å. We are now in a position to analyse these structures and to perform detailed calculations to learn how the mutational effects are implemented. This work was performed by three graduate and three undergraduate students, in addition to senior personnel. The technical and scientific training was broad-based and sophisticated and has prepared them well for future careers and career choices.
