Assembly and Function of the Cyanobacterial H2O-Oxidation Complex

Oklahoma State University, Stillwater OK

Investigators

Abstract

Oxygenic photosynthesis is ultimately the source of the vast majority of biological energy that supports life on earth and is responsible for the generation of oxygen strictly required for respiration in all animals, including humans. Oxygenic photosynthesis depends upon a cluster of four manganese atoms, (Mn)4, associated with the catalytic site of H 2 O-oxidation of the membrane-bound photosystem II (PSII) complex. Recent advances in the determination of the molecular structure confirms that the (Mn)4) is sequestered within a region of the PSII complex formed by intrinsic and extrinsic proteins near the lumenal surface of the thylakoid membrane. Many outstanding questions remain and their mystery, in some cases, is only deepened by the newly available structure. The overall objective of this project is to connect the structure features of the cyanobacterial PSII H2O-oxidation complex (WOC) to the dynamical aspects of the assembly and function of the Mn cluster. Cyanobacteria are being used as the experimental model since the photosynthetic mechanism in these ubiquitous organisms is fundamentally similar to that found in higher plants and algae, but at the same time, more efficient methods for probing the photosynthetic process at the molecular level are available thereby shortening the time necessary to test the basic ideas of the research. The work combines molecular genetic, biochemical and biophysical approaches to clarify the process of photoactivation, which is the sequential light- dependent assembly of the catalytic tetramer of Mn atoms that forms the core of the WOC. The research is facilitated by the development of His-tag purification methods and strains for the gentle isolation of highly active and pure detergent-solublized PSII complexes. The ability to produce large amounts of mutant and wild-type PSII particles and extrinsic proteins now permits systematic manipulations of the ions and proteins of the WOC to better understand its assembly and function. While most mutants are already produced, a few additional mutations are being made to perform site-specific labeling to probe structural rearrangements using a cysteine-scanning mutagenesis/chemical modification approach. The experiments are of benefit to parallel biophysical analyses (e.g. FTIR, EPR) by defining the compositional and kinetic properties of specific mutant His-tagged PSII particles and highlighting the best preparative procedures for isolating and analyzing them. Together, these efforts complement on-going developments in the crystal structure determination by considering the dynamical aspects concerning the mechanism of light-driven assembly and activation of the Mn cluster.