Our research focuses on the catalytic production of oxygen by photosystem II (PSII) of higher plants. The evolution of O 2 occurs during the conversion of solar energy into a chemical form that is usable by all living things and in addition provides the O 2 required by respiring life forms. PSII contains a Mn 4Ca ion cluster at the site where O 2 is produced from H 2O. We are particularly interested in how chloride functions in the activation of catalysis in this system. 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 Research Corporation, the Dreyfus Foundation, and the National Science Foundation.
Photosystem II is a large integral-membrane protein complex located in the thylakoid membrane of chloroplasts. In addition to the Mn 4Ca cluster, which cycles through five oxidation states during catalysis, the site of oxygen evolution includes a redox active tyrosine residue (Tyr Z) and Cl -. Although much is known about PSII at a molecular level, thanks to recent X-ray crystallography studies at 3.0-3.8 Å resolution, the mechanism by which H 2O is converted into O 2 is not well understood. For example, while the Mn ions are known generally to be in the 3+ or 4+ state, the exact oxidation states of the various Mn ions during the catalytic cycle are not known. Similarly, while Cl - is required for O 2 evolution, it has not been resolved in the X-ray crystal structures of PSII. Its exact role is not known, but it is evidently related to proton transfer events and/or charge neutralization during successive oxidation steps. A better understanding of the role of Cl - in PSII may help reveal its role in other Cl --binding enzymes, such as cytochrome oxidase and a -amylase.
We have carried out studies of Cl - function using anions that act as activators or inhibitors. Using an O 2 sensitive electrode to measure the activity of PSII, the characteristics of an anion can be analyzed using steady state kinetics. Anions besides Cl - that can activate oxygen evolution include Br -, NO 3 -, and I -. Ions that act as inhibitors of oxygen evolution include F -, N 3 ‑, and I -. EPR 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 Mn 4Ca cluster of PSII and its nearby tyrosine radical, Tyr Z, as well as from other electron transfer center. These signals give a direct indication of the oxidation state of the Mn 4Ca cluster. In addition, they are sensitive to the nearby environment and can indicate changes in ligation or the protonation state of nearby amino acids. The EPR signals are therefore sensitive to the presence of Cl - or other anions and can reveal molecular changes that are related to the behavior found in enzyme kinetics studies.
Haddy, A. "EPR Spectroscopy of the Manganese Cluster of Photosystem II " (Review) Photosynthesis Research 2007 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 S 2 state of Photosystem II confirms an S=5/2 origin of the X-band g=4.1 signal "Biophysical Journal 2004 87, 2885-2896
Benson, D.E.; Haddy, A.E.; Hellinga, H.W. "Converting a Maltose Receptor into a Nascent Binuclear Copper Oxygenase by Computational Design "Biochemistry 2002 41, 3262-3269
Haddy, A. "Using a Molecular Modeling Program to Calculate Electron Paramagnetic Resonance Hyperfine Couplings in Semiquinone Anion Radicals "Journal of Chemical Education 2001 78, 1206-1208
Haddy, A. ; Kimel, R.A.; Thomas, R. "Effects of Azide on the S 2 State EPR Signals from Photosystem II "Photosynthesis Research 2000 63, 35-45
Haddy, A.; Smith, G. "Paramagnetic Components of Carp Liver Tissue Observed by Electron Paramagnetic Resonance Spectroscopy "Comparative Biochemistry and Physiology, Part B 1999 123, 407-415
Haddy, A.; Hatchell, J.A.; Kimel, R.A.; Thomas, R. "Azide as a Competitor of Chloride in Oxygen Evolution by Photosystem II "Biochemistry 1999 38, 6104-6110