ORCID

https://orcid.org/0000-0002-6213-7040

Date of Award

Winter 12-15-2016

Author's School

Graduate School of Arts and Sciences

Author's Department

Chemistry

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Photosystem II (PSII) is a protein complex found embedded in the thylakoid membranes of all organisms that perform oxygenic photosynthesis. PSII converts sunlight into chemical energy, filling our atmosphere with molecular oxygen in the process and supporting nearly all life on Earth. PSII undergoes frequent light-induced damage as an unavoidable result of the electron transfer reactions it catalyzes. When damaged, PSII is disassembled, repaired, and reassembled in an intricate, tightly regulated process. The structure and mechanism of function of active PSII are relatively well-understood, due to the available crystal structures of the active complex and many years of biochemical and biophysical investigation. However, many aspects of the broader PSII life-cycle are less clear. In this work, several structural aspects of the PSII life-cycle are investigated, with an approach that emphasizes mass spectrometry (MS)-based tools. The field of protein MS is developing rapidly, and, especially in the last several years, MS has become a key tool for addressing a variety of questions in the area of photosynthesis. Chapter 1 provides an in-depth review of the ways in which MS has been, and can be, applied to PSII life-cycle research. This chapter presents the relevant MS-based techniques, as well as the knowledge that has been gained about the PSII life-cycle through their application. The work in Chapter 2 used cross-linking and MS to identify the binding site of the Psb28 protein to PSII. Psb28 binds transiently to a PSII assembly intermediate complex, exerting a protective effect on this complex. However, since Psb28 dissociates before assembly is complete, it is not found in the crystal structure and its structural location within the complex has remained unknown. We used isotope-encoded chemical cross-linking followed by MS to identify the binding partners of Psb28 in the model cyanobacterium Synechocystis sp. PCC 6803, the organism used throughout this work. We identified three cross-links between Psb28 and the α- and β- subunits of cytochrome b559 (PsbE and PsbF), pinpointing the structural location of Psb28 on the cytosolic surface of PSII in close association with these subunits. Our results allow us to propose several mechanisms by which Psb28 could exert its protective effect. In Chapter 3, we used high-resolution tandem MS to identify oxidative modifications in PSII. We found that the total number of modified residues increased by over 50% following light incubation, with the D1 protein showing the most marked increase (3.3-fold) of the proteins we monitored. These results strongly support the idea that ROS are generated as a byproduct of PSII photochemistry and that they damage PSII subunits, especially D1, which has the fastest turnover rate of all the subunits. By mapping the modified residues onto the PSII crystal structure, we found that the lumen-side residues form two nearly continuous, roughly linear "arms" starting at the Mn4Ca cluster and radiating outward all the way to the surface of PSII. We propose that these two "arms" are oxygen/ROS exit channels that protect PSII by removing ROS from the complex after they are generated at the Mn4Ca cluster. It has long been believed that PSII must contain such channels, and this study provides the most complete and descriptive molecular-level evidence yet for their existence and location. Chapter 4 describes a study that used cross-linking and MS to identify the binding location of PsbQ on the lumenal surface of PSII. Though PsbQ is a necessary component of PSII complexes with highest oxygen-evolving activity, it is not found in the available cyanobacterial crystal structures. Our results show that PsbQ helps stabilize the PSII dimer, providing a structural basis to explain our biochemical data and previous findings. A novel PSII subcomplex with multiple copies of the PsbQ protein was also discovered, and its characterization is described in this chapter. Based on our results, we propose it is a late PSII assembly intermediate that stabilizes the active PSII dimer just before association of the other lumenal extrinsic proteins PsbU and PsbV.

Language

English (en)

Chair and Committee

Michael L. Gross, Himadri Pakrasi

Committee Members

Robert E. Blankenship, J. Dewey Holten, Joseph M. Jez

Comments

Permanent URL: https://doi.org/10.7936/K7X63KBB

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