Oxygen evolution by photosystem II: The Cl- and Ca2+ requirements
Our research focuses on the catalytic production of oxygen by photosystem II (PSII) of higher plants. The evolution of O2 occurs during the conversion of solar energy into chemical energy, a process that is promoted by the absorption of light by photosystem I and photosystem II within the thylakoid membranes of chloroplasts. Ultimately the absorption of light results in the synthesis of carbohydrates, which are consumed by both plants and animals. In addition, the reactions that take place at photosystem II provide the O2 that is used by respiring life forms. O2 is produced at a catalytic Mn4Ca cluster, which uses H2O as the initial source of electrons in the electron transfer process associated with the conversion of light energy. This complex catalytic reaction is activated by chloride ions. In our laboratory, we are interested in how the Ca2+ ion of the Mn4Ca cluster and the activating Cl- ion function in the O2 evolution process. To carry out this research, we combine biochemical techniques such as protein purification and enzyme kinetics assays with physical techniques, particularly electron paramagnetic resonance (EPR) spectroscopic. Projects in our lab have been funded by the National Science Foundation, the Dreyfus Foundation, and the Research Corporation.
Photosystem II is a large integral-membrane protein complex located in the thylakoid membrane of chloroplasts. At its center PSII contains the light-absorbing reaction center, a cluster of chlorophyll molecules at which primary charge separation occurs after it has entered an excited state. The charge separation within PSII, along with a similar event within photosystem I, promotes electron transfer through numerous redox active centers, eventually producing the transmembrane proton gradient that is required for ATP synthesis. At the site of oxygen evolution, where electron transfer begins, the Mn4Ca cluster cycles through five oxidation states during catalytic production of O2. In addition, the site includes a redox active tyrosine residue (Tyr Z), other coordinating amino acid residues, and one or more nearby Cl- ion. Although much is known about the structure of PSII, thanks to recent X-ray crystallography studies at 3.0-3.8 Å resolution, the mechanism by which H2O is converted into O2 is not well understood. For example, while the Mn ions are known generally to be in the 3+ or 4+ state, their exact oxidation states and couplings throughout the catalytic cycle are not known. The directly bound Ca2+ ion and the nearby Cl- ion have important roles in coordinating the step-wise oxidation of the Mn ions with the transfer of electrons from H2O, but this is poorly understood. The Ca2+ ion, although somewhat labile, appears to have a critical role in electron transfer to the Tyr Z residue. The Cl- ion, although more distantly bound, is also required for electron transfer in the higher oxidation states. Both ions may participate in proton movement or a hydrogen bond network, function in charge neutralization, influence the coupling of the Mn4Ca cluster, or a combination of these possibilities.
Electron paramagnetic resonance spectroscopy detects transitions between electron spin levels of unpaired electrons and is sensitive to the chemical environment of the paramagnetic center. EPR signals are observed from the Mn4Ca cluster and the nearby tyrosine radical, Tyr Z, as well as from other electron transfer centers of PSII. The signals from the Mn4Ca cluster give a direct indication of its oxidation state and coupling. In addition, the signals can reveal changes in ligation to the Ca2+ ion or alterations in coordinating residues. Using enzyme kinetics studies, we have characterized the function of Cl- in PSII by replacing it with anions that can activate or inhibit O2 evolution. Studies of inhibition kinetics have characterized fluoride and azide anions, both weak bases, as competitive inhibitors of chloride activation. Iodide has been found to be an activator that shows substrate inhibition at higher concentrations. Other anions, such as bromide and nitrate, are primarily activators of O2 evolution. Our more recent studies have examined the Ca2+ requirement of PSII, including the effects of decreased pH and elevated NaCl concentrations on Ca2+ ligation to the Mn cluster. By combining enzyme kinetics analyses with EPR spectroscopy, the effects of these inorganic ions are understood at a more molecular level. These studies have the eventual goal of leading to a better understanding of how these ions affect the function of the Mn4Ca cluster in the catalytic cycle that produces O2.
Haddy, A.; Ore, B.M. "An alternative method for calcium depletion of the oxygen evolving complex of photosystem II as revealed by the dark-stable multiline EPR signal" Biochemistry 2010 49, 3805-3814
Kuntzleman, T; Haddy, A. "Fluoride Inhibition of Photosystem II and the Effect of Removal of the PsbQ Subunit"Photosynthesis Research 2009 102, 7-19
Haddy, A. "EPR Spectroscopy of the Manganese Cluster of Photosystem II" (Review) Photosynthesis Research2007 92, 357-368
Bryson, D.I.; Doctor, N.; Johnson, R.; Baranov, S.; Haddy, A. "Characteristics of Iodide Activation and Inhibition of Oxygen Evolution by Photosystem II" Biochemistry 2005 44, 7354-7360
Haddy, A.; Lakshmi, K.V.; Brudvig, G.W.; Frank, H.A. "Q-band EPR of the S2 state of Photosystem II confirms an S=5/2 origin of the X-band g=4.1 signal" Biophysical Journal 2004 87, 2885-2896