Assembly and Function of the Cyanobacterial Photosystem II Complex

Oklahoma State University, Stillwater OK

Investigators

Abstract

Intellectual Merit: Oxygenic photosynthesis is the major solar energy conversion process in the biosphere consequently it is at the very foundation of life on planet Earth. Photosystem II (PSII) may be considered to be the key enzyme of this process since it uses light energy to split water (water oxidation) liberating the tightly bound hydrogens from the oxygen of water and, at the same time, renders these hydrogens in a form used for producing energy-rich organic carbon compounds from inorganic carbon (carbon dioxide). This project investigates the water-oxidation enzyme of PSII, the replacement of damaged PSII proteins, and assembly of Mn and Ca into the active site. PSII is a dynamic structure that is under a constant state of repair owing to the incessant photodamage it incurs during its normal operation. The repair occurs via a complex assembly pathway involving the removal of damaged D1 protein, insertion and proteolytic processing of the nascent D1 protein precursor and the assembly of the metal atoms of the manganese cluster (4Mn-Ca), which is the heart of the water oxidation reaction. The D1 protein is the locus of most of the above mentioned photodamage. A dedicated and conserved D1 damage detection and replacement mechanism has evolved to repair the damage, but remains to be understood. 1. Mechanism of 4Mn-Ca assembly. The active site of water-oxidation contains a cluster of four manganese and one calcium ions (4Mn-Ca) most of which are bound by the labile D1 protein. The assembly of the 4Mn-Ca requires light to drive the assembly into an active metal cluster. For reasons that remain unclear, this assembly process occurs with very low quantum efficiency. Previous NSF-supported analysis revealed new kinetic features of this process. Hypotheses regarding the origin of these new kinetic features will be evaluated using site-directed mutagenesis together with sensitive biochemical and biophysical techniques that follow the assembly process. The metalloprotein assembly process is being considered in a kinetic framework, that if valid, would represent a new, stochastic concept of metal cluster assembly. 2. Detection of structural changes during the removal and replacement of damaged D1 protein during the PSII repair cycle. The D1 protein is buried deeply within the large PSII complex and, consequently, major rearrangements of the PSII complex must occur during the replacement of damaged D1 and the insertion and activation of a new copy of the protein. How is internal damage within the PSII complex detected and what are the signals to initiate repair? What is happening to the D1 protein during replacement? What are the structural changes associated with the assembly of the metal cluster? Two complementary approaches to experimentally address these questions are used: targeted structure-reactivity approaches using mutant strains containing non-native cysteines that will be reacted with thiol-reactive reagents to quantitatively probe site-specific changes in surface exposure. This directed approach will be complemented using powerful mass spectroscopic methods which have the potential to discover assembly associated changes in exposure at other locations in the protein. Broader Impacts: Educational impacts include student exposure to a multidisciplinary attack on an interesting biological problem and training to utilize, operate, and understand advanced mass spectrometers. An education collaboration with the Native Americans in Biological Sciences (NABS) program is planned. The project incorporates a teaching module tentatively entitled: "Light and Life". It will contain exercises and materials for the "Biological Dilemma" (a multi-faceted real-world issue) and for secondary school teachers to utilize in their home science classes. These will borrow materials from the research project and be modeled on the most successful activities of earlier programs, which involved close collaboration with the high school teachers visiting the campus during the summer academies.