Gary W. Brudvig
Department of Chemistry
P.O. Box 208107
New Haven, CT 06520-8107
1983 Searle Scholar
What is the molecular basis for energy transduction in plant photosynthesis? This is the question that has been the focus of our research program. The first step in the energy-transduction process is the light- induced charge-separation reaction that occurs in a membrane-protein complex called the photoreaction center. Thereafter, a series of rapid electron-transfer reactions serve to stabilize the charge-separated state. Later still, the photochemically-produced oxidant and reductant are consumed in the oxidation of water and the reduction of carbon dioxide. One of the primary targets of our research is the plant enzyme called photosystem II that catalyzes the oxidation of water to dioxygen at a site within the enzyme containing a tetranuclear manganese cluster. Photosystem II is one of two reaction center complexes that initiate the light-driven electron transfer reactions of plant photosynthesis. The long- term objective of our research is to develop an understanding at the molecular level of the conversion of light energy into chemical energy in plant photosynthesis. Toward this goal, we are pursuing several related lines of research. A major effort involves biophysical studies of the purified photosystem II complex itself. We use spectroscopic and biophysical methods to probe the structure of the redox centers, the kinetics and yields of electron-transfer reactions, and the chemistry of water oxidation in photosystem II. Our aim is to define how Nature has solved the difficult problem of the efficient light-driven, four-electron oxidation of water to O2. It is hoped that these studies will provide insight into the design of artificial systems that split water. Toward this goal, we are also investigating inorganic models of the manganese site in photosystem II. Because the model complexes are more easily characterized, the inorganic studies provide important information that can aid the interpretation of results from the biological system. On the other hand, the information from the biophysical studies better define the nature of the catalytic manganese complex that is to be modeled. The synergism between the inorganic and biological chemistry is an important aspect of this research. A third area of research involves the use pulsed EPR spectroscopy to study the magnetic interactions between paramagnetic sites in proteins. These studies are aimed at determining the distances between redox centers in electron-transfer proteins and the magnetic properties of metal ion clusters in metalloproteins. Some of the proteins that we are investigating include photosystem II, photosystem I, the photoreaction center protein from Rb. sphaeroides, ribonucleotide reductase, nitric oxide synthase and cytochrome c oxidase.
Biophysical Studies of Photosystem II
We use EPR and optical spectroscopy to monitor the photochemical events and to obtain structural and mechanistic information on photosystem II. Photosystem II contains more than ten distinct redox- active centers. One of these is the tetranuclear manganese cluster that catalyzes the oxidation of water. We study photosystem II samples that are prepared and trapped at low temperature in each of the oxidation states of the manganese complex. The binding and reactions of substrates are also studied in order to define the structure and chemical properties of the manganese cluster as it proceeds through the catalytic cycle. In order to control the turnover of photosystem II in the highly-concentrated samples needed for spectroscopic studies, we have used herbicides that bind to photosystem II and block electron transfer. One recent extension of this approach involves tethering a redox-active center to the herbicide so that multi-electron turnover control can be achieved. Other projects involve the use of optical or fluorescence spectroscopy to study the electron-transfer reactions that occur during the water- oxidation cycle. Photosystem II contains several redox centers, including cytochrome b559, that do not play a direct role in the reactions leading to water oxidation. The function of these species is currently not well understood, but they may be involved in protection of photosystem II from photodamage. By using a combination of spectroscopic measurements, functional assays and site-directed mutagenesis, we are working to reveal how these alternate electron donors function.
Inorganic Models of the Manganese Cluster in Photosystem II
The S states in the water-oxidation cycle are different oxidation states of the tetranuclear manganese cluster. The chemistry of this manganese cluster involves high-valent manganese and H2O. The goal of inorganic modeling studies is to give an insight into the high-valent Mn chemistry in aqueous media that may be relevant to the photoactivated assembly of the tetranuclear manganese cluster, its structure and physical properties, and the mechanism of water oxidation. To date, the syntheses of high-valent manganese complexes have relied on self-assembly methods. We are now using molecular modeling to aid in the synthesis of high-valent manganese model complexes. EPR spectroscopy, magnetics and electrochemistry are some of the methods that we use to characterize the manganese complexes.
Determining Distances by Pulsed EPR
Michel, Deisenhofer and Huber, won the Nobel Prize in chemistry for their solution of the crystal structure of the bacterial photoreaction center, a light-driven, electron-transport enzyme which is believed to have some structural homology to the "core" polypeptides of photosystem II. However, a crystal structure of photosystem II is not available and few distance/structural measurements have been made. We have developed a theoretical analysis of electron spin-lattice relaxation transients to obtain the magnitude of the exchange and dipolar interactions between paramagnetic sites within an enzyme. The magnitude of the dipolar interaction is a reflection of the distance between two sites and the magnitude of the exchange interaction is related to the rate of electron transfer between two sites. These factors are of central importance in order to understand the relations between distance and rate of long-range electron transfer in biological systems. Recent work on photosystem II has been aimed at determining the locations of the two redox-active tyrosines. Photosystem II has a non-heme Fe(II) located between the quinones QA and QB. We have used electron spin-lattice relaxation measurements on photosystem II to estimate the distance between the non-heme Fe(II) and the tyrosine electron donors: YD and YZ. We find that the Fe(II)-YD and Fe(II)-YZ distances are both approximately 37 which agree well with those predicted from the structure of the bacterial photoreaction center.
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