The overall goal is to understand the operation of metal-ion clusters and electron transfer processes in biological systems. The specific goal is to understand the mechanism of photosynthetic water oxidation. Oxygenic photosynthesis provides the molecular oxygen and fixed carbon required to sustain animal life. The proposed work will identify the ligands to the manganese and calcium ions located at the catalytic site of photosynthetic water oxidation. Identification of these ligands will impose new constraints on models for the operation of the manganese cluster, and will enable such models to be placed in the context of protein structure. The information obtained will be applicable to other metal-ion clusters, and to the study of membrane proteins and biological electron transfer processes in general. Electron transfer can be studied more easily and precisely in photosynthetic than in mitochondrial systems, because electron transfer can be conveniently initiated with light and studied under single turn-over conditions. In oxygenic photosynthesis, Photosystem II (PSII) uses light to extract electrons from water and donate them into an electron transport chain that generates the chemical free energy and reducing equivalents required for carbon fixation. PSII photochemistry takes place in a heterodimer of two polypeptides known as D1 and D2. A cluster of four manganese ions accumulates four oxidizing equivalents in response to this photochemistry, and then uses them to oxidize two molecules of water in concerted mechanism that requires calcium and releases one molecule of oxygen as a by-product. The ligands for both manganese and calcium are believed to be predominantly carboxyl residues on the D1 and D2 polypeptides. The proposed work will identify the specific carboxyl ligands to manganese and calcium, and characterize their influence on oxygen evolution. This will be accomplished by site-directed mutagenesis of the psbA and psbD genes from the unicellular cyanobacterium Synechocystis sp. PCC 6803, which encode the D1 and D2 polypeptides, respectively. A mutagenic screening procedure will rapidly identify those carboxyl residues most likely to serve as ligands. Mutations of these residues will be characterized by biochemical and spectroscopic methods, while the remaining mutants will be archived for future analysis. I have previously employed site-directed mutagenesis of the psbA and psbD genes of Synechocystis sp. PCC 6803 to identify the two redox-active tyrosine residues in the PSII core that interact with the manganese cluster.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM043496-05
Application #
2182035
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1989-12-01
Project End
1995-06-30
Budget Start
1993-12-01
Budget End
1995-06-30
Support Year
5
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of California Riverside
Department
Biochemistry
Type
Schools of Earth Sciences/Natur
DUNS #
City
Riverside
State
CA
Country
United States
Zip Code
92521
Debus, R J; Campbell, K A; Gregor, W et al. (2001) Does histidine 332 of the D1 polypeptide ligate the manganese cluster in photosystem II? An electron spin echo envelope modulation study. Biochemistry 40:3690-9
Debus, R J (2001) Amino acid residues that modulate the properties of tyrosine Y(Z) and the manganese cluster in the water oxidizing complex of photosystem II. Biochim Biophys Acta 1503:164-86
Chu, H A; Debus, R J; Babcock, G T (2001) D1-Asp170 is structurally coupled to the oxygen evolving complex in photosystem II as revealed by light-induced Fourier transform infrared difference spectroscopy. Biochemistry 40:2312-6
Faller, P; Debus, R J; Brettel, K et al. (2001) Rapid formation of the stable tyrosyl radical in photosystem II. Proc Natl Acad Sci U S A 98:14368-73
Debus, R J; Campbell, K A; Pham, D P et al. (2000) Glutamate 189 of the D1 polypeptide modulates the magnetic and redox properties of the manganese cluster and tyrosine Y(Z) in photosystem II. Biochemistry 39:6275-87
Debus, R J; Campbell, K A; Peloquin, J M et al. (2000) Histidine 332 of the D1 polypeptide modulates the magnetic and redox properties of the manganese cluster and tyrosine Y(Z) in photosystem II. Biochemistry 39:470-8
Debus, R J (2000) The polypeptides of photosystem II and their influence on manganotyrosyl-based oxygen evolution. Met Ions Biol Syst 37:657-711
Qian, M; Dao, L; Debus, R J et al. (1999) Impact of mutations within the putative Ca2+-binding lumenal interhelical a-b loop of the photosystem II D1 protein on the kinetics of photoactivation and H2O-oxidation in Synechocystis sp. PCC6803. Biochemistry 38:6070-81
Hays, A M; Vassiliev, I R; Golbeck, J H et al. (1999) Role of D1-His190 in the proton-coupled oxidation of tyrosine YZ in manganese-depleted photosystem II. Biochemistry 38:11851-65
Hundelt, M; Hays, A M; Debus, R J et al. (1998) Oxygenic photosystem II: the mutation D1-D61N in Synechocystis sp. PCC 6803 retards S-state transitions without affecting electron transfer from YZ to P680+. Biochemistry 37:14450-6

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