Debus, Richard J.
Professor of Biochemistry

Physical Biochemistry Plant Biochemistry and Molecular Biology Ph.D., University of California, San Diego, 1985 McKnight Foundation Postdoctoral Fellow in Photosynthesis, 1985-1986 NSF Postdoctoral Fellow in Plant Biology, 1986-1988 951-827-3483 richard.debus@ucr.edu

My laboratory's overall goal is to determine how plants use sunlight to convert water to molecular oxygen. The catalytic site is located in photosystem II near the lumenal surface of the thylakoid membrane. Photosystem II is an integral membrane protein complex consisting of nearly 30 different polypeptides and over 50 organic and inorganic cofactors. The catalytic site contains a Mn₄Ca cluster that interacts closely with a redox-active tyrosine residue known as YZ. The absorption of light leads to the oxidation of YZ, forming the YZ^• radical. The YZ^• radical extracts electrons and protons from the Mn₄Ca cluster, leading to the oxidation of water and the release of O2. The extracted protons are deposited in the thylakoid lumen, contributing to the transmembrane pH gradient and the synthesis of ATP. Photosynthetic water oxidation takes place under severe energetic and mechanistic constraints and is tightly controlled by the protein environment of the Mn₄Ca-YZ complex. This control matches the potential of the Mn₄Ca cluster to the potential of the YZ^• radical, even as the Mn₄Ca cluster accumulates oxidizing equivalents, and prevents the release of toxic, partly oxidized intermediates. Our goal is to identify the amino acid residues that are responsible for this control and to determine the role of each. Our strategy is to combine site-directed mutagenesis and isotopic labeling with spectroscopic characterizations involving Fourier-transform infrared (FTIR), electron paramagnetic resonance (EPR), and time-resolved optical absorption and fluorescence spectroscopies. Our recent efforts have shown that the C-terminus of the D1 polypeptide ligates the Mn ion that undergoes oxidation during the first step in the catalytic cycle, and that the Mn ion recently identified as the "most catalytically active" paradoxically does not undergo oxidation during the first three steps of the catalytic cycle.

Photosystem II provides the O2 we breathe and the electrons and protons that ultimately provide the food we eat. Developing a detailed mechanistic description of water oxidation by photosystem II remains one of the major goals of bioenergetics. Understanding this process should provide insight into the design of synthetic catalysts for the commercial production of O2 and H2 and provide insight into the mechanisms of two important classes of enzymes that are currently the subjects of intensive bio-medical investigations: metalloradical enzymes and enzymes whose mechanisms involve proton-coupled electron transfer reactions. Such enzymes catalyze biological energy transduction in mitochrondria. Elucidating the catalytic mechanisms of these enzymes is essential for understanding the molecular basis of mitochondrial diseases and aging. Photosystem II is both an excellent example of a metalloradical enzyme and a unique laboratory for studying proton-coupled electron transfer reactions. Its advantages derive from its ability to be stepped through its catalytic cycle with single flashes of light, thereby facilitating studies of reaction cycle intermediates.

The primary organism we employ for our studies is the unicellular cyanobacterium, Synechocystis sp. strain PCC 6803. This organism has essentially the same photosynthetic apparatus as higher plants but its complete genome sequence is available, it can be manipulated genetically more easily than higher plants, and it can be propagated without need for photosystem II. The latter trait permits the study of mutations that would be lethal to most photosynthetic organisms.

SELECTED PUBLICATIONS

Debus, R. J. (2008) Protein Ligation of the Photosynthetic Oxygen-Evolving Center, Coord. Chem. Rev. 252, 244-258.

Singh, S., Debus, R. J., Wydrzynski, T., and Hillier, W. (2008) Investigation of Substrate Water Interactions at the High Affinity Mn Site in the Photosystem II Oxygen Evolving Complex, Phil. Trans. R. Soc. B 363, 1229-1235.

Strickler, M. A., Hwang, H. J., Burnap, R. L., Yano, J., Walker, L. M., Service, R. J., Britt, R. D., Hillier, W. and Debus, R. J. (2008) Glutamate-354 of the CP43 Polypeptide Interacts with the Oxgyen-Evolving Mn₄Ca Cluster of Photosystem II: A Preliminary Characterization of the Glu354Gln Mutant, Phil. Trans. R. Soc. B 363, 1179-1188.

Yeagle, G. J., Gilchrist, M. L., Jr., Walker, L. M., Debus, R. J., and Britt, R. D. (2008) Multifrequency Electron Spin Echo Envelope Modulation Studies of Nitrogen Ligation to the Manganese Cluster of Photosystem II, Phil. Trans. R. Soc. B 363, 1157-1166.

Hwang, H. J., McLain, A., Debus, R. J., and Burnap, R. L. (2007) Photoassembly of the Manganese Cluster in Mutants Perturbed in the High Affinity Mn-Binding Site of the H₂O-Oxidation Complex of Photosystem II, Biochemistry 46, 13648-13657.

Hwang, H. J., Dilbeck, P., Debus, R. J., Burnap, R. L. (2007) Mutation of Arginine-357 of the CP43 Protein of Photosystem II Severely Impairs the Catalytic S-state Cycle of the H₂O-Oxidation Complex, Biochemistry 46, 11987-11997.

Strickler, M. A., Walker, L. M., Hillier, W., Britt, R. D., and Debus, R. J. (2007) No Evidence from FTIR Difference Spectroscopy that Aspartate-342 of the D1 Polypeptide Ligates a Mn Ion that Undergoes Oxidation During the S0 to S1, S1 to S2, or S2 to S3 Transitions in Photosystem II, Biochemistry 46, 3151-3160.

Strickler, M. A., Hillier, W., and Debus, R. J. (2006) No Evidence from FTIR Difference Spectroscopy that Glutamate-189 of the D1 Polypeptide Ligates a Mn Ion that Undergoes Oxidation During the S0 to S1, S1 to S2, or S2 to S3 Transitions in Photosystem II, Biochemistry 45, 8801-8811.

Strickler, M. A., Walker, L. M., Hiller, W. and Debus, R. J. (2005) Evidence from Biosynthetically Incorporated Strontium and FTIR Difference Spectroscopy that The C-Terminus of the D1 Polypeptide of Photosystem II Does Not Ligate Calcium, Biochemistry 44, 8571-8577.

Debus, R. J., Strickler, M. A., Walker, L. M. and Hiller, W. (2005) No Evidence from FTIR Difference Spectroscopy that Aspartate-170 of the D1 Polypeptide Ligates a Manganese Ion that Undergoes Oxidation during the S0 to S1, S1 to S2, or S2 to S3 Transitions in Photosystem II, Biochemistry 44, 1367-1374.

Chu, H.-A., Hiller, W., and Debus, R. J. (2004) Evidence that the C-Terminus of the D1 Polypeptide of Photosystem II is Ligated to the Manganese Ion that Undergoes Oxidation During the S1 to S2 Transition: An Isotope-Edited FTIR Study, Biochemistry 43, 3152-3166.

Clausen, J., Debus, R. J., and Junge, W. (2004) "Time-Resolved Oxygen Production by Photosystem II: Chasing Chemical Intermediates," Biochim. Biophys. Acta (Bioenergetics) 1655, 184-194.

Debus, R. J., Aznar, C., Cambpell, K. A., Gregor, W., Diner, B. A., and Britt, R. D. (2003) "Does Aspartate 170 of the D1 Polypeptide Ligate the Manganese Cluster in Photosystem II? An EPR and ESEEM Study," Biochemistry 42, 10600-10608.

Faller, P., Rutherford, A. W., and Debus, R. J. (2002) "Tyrosine D Oxidation at Cryogenic Temperature in Photosystem II," Biochemistry 41, 12914-12920.

Faller, P., Debus, R. J., Brettel, K., Sugiura, M., Rutherford, A. W., and Boussac, A. (2001) "Rapid Formation of the Stable Tyrosyl Radical in Photosystem II," Proc. Natl. Acad. Sci. (USA) 98, 14368-14373.

Debus, R. J., Campbell, K. A., Gregor, W., Li, Z.-L., Burnap, R. L., and Britt, R. D. (2001) "Does Histidine 332 of the D1 Polypeptide Ligate the Manganese Cluster in Photosystem II? An Electron Spin Echo Envelope Modulation Study," Biochemistry 40, 3690-3699.

Chu, H.-A., Debus, R. J., and 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, 2312-2316.

Debus, R. J. (2001) "Amino Acid Residues that Modulate the Properties of Tyrosine YZ and the Manganese Cluster in the Water-Oxidizing Complex of Photosystem II," Biochim. Biophys. Acta (Bioenergetics) 1503, 164-186.

Hays, A.-M. A., Vassiliev, I. R., Golbeck, J. H. and Debus, R. J. (1999) "Role of D1-His190 in the Proton-Coupled Oxidation of Tyrosine YZ in Manganese-Depleted Photosystem II," Biochemistry 38, 11851-11865.

Hays, A.-M. A., Vassiliev, I. R., Golbeck, J. H. and Debus, R. J. (1998) "Role of D1-His190 in Proton-Coupled Electron Transfer Reactions in Photosystem II: A Chemical Complementation Study," Biochemistry 37, 11352-11365.

Campbell, K. A., Peloquin, J. M., Pham, D. P., Debus, R. J. and Britt, R. D. (1998) "Parallel Polarization EPR Detection of an S1-State 'Multiline' EPR Signal in Photosystem II Particles from Synechocystis sp. PCC 6803," J. Am. Chem. Soc. 120, 447-448.

Chu, H.-A., Nguyen, A. P. and Debus, R. J. (1995) "Amino Acid Residues that Influence the Binding of Manganese or Calcium to Photosystem II. 2. The Carboxy-Terminal Domain of the D1 Polypeptide," Biochemistry 34, 5859-5882.

Chu, H.-A., Nguyen, A. P. and Debus, R. J. (1995) Amino acid Residues that Influence the Binding of Manganese or Calcium to Photosystem II. 1. The Lumenal Interhelical Domains of the D1 Polypeptide," Biochemistry 34, 5839-5858.

Debus, R. J. (1992) "The Manganese and Calcium ions of Photosynthetic Oxygen Evolution," Biochim. Biophys. Acta (Bioenergetics) 1102, 269-352.