Photosystem II as a model protein for understanding metalloenzyme active site assembly

Project Summary Metalloenzymes are ubiquitous throughout biology and implicated in a variety of human diseases. A gap in our understanding of metalloenzymes lies in their maturation. Biogenesis intermediates precede formation of the mature enzyme and must be tightly regulated to correctly assemble the metallocofactor in the active site. Despite the wealth of knowledge on mature metalloenzymes, their biogenesis, especially active site assembly, is not well understood. Photosystem II (PSII) is a light-driven oxidoreductase whose active site contains a metallocofactor that is assembled through a series of step-wise ion binding and photooxidation events called photoactivation. Photoactivation provides a convenient model for understanding metallocofactor assembly because it is facile; in vitro assembly may be achieved by simply adding the required ions in solution and assembly is advanced by providing light quanta. Furthermore, recent advances in structural investigation of PSII have developed a platform for routinely solving high-resolution structures of PSII assembly intermediates. We propose to characterize biogenesis intermediates of PSII that may reveal generalized rules for metalloenzyme assembly. The long-term goals of this project are to provide structural and functional bases for assembly of the metallocofactor in PSII (training phase), and reveal how PSII biogenesis intermediates maintain precursor structures important for active site maintenance (independent phase). We will use structural approaches, biochemical techniques, and computational modeling to achieve the following three specific objectives: (1) to reveal how the oxygen evolving complex of PSII is assembled, (2) to uncover precursor structures of PSII that are integral for assembling its active site, and (3) to understand the role of transiently-bound subunits during PSII biogenesis. Gaining such insight may allow for the development of new therapeutics and design principles for de novo metalloenzyme engineering.