Structure and Function of the Fmo Protein from the Photosynthetic Green Sulfur Bacteria

Photosynthesis is a central biological process that produces all our food and the majority of energy used by human beings. Intense attention has been focused on using photosynthetic organisms or mechanisms adapted from photosynthesis as sources to produce cheap, clean and renewable energy. A deep understanding of the molecular mechanism of the photosynthetic processes is essential as part of that effort. Photosynthetic prokaryotes called green sulfur bacteria (GSB) have been used as model species to understand the mechanism of the energy capture and storage and the molecular structures of the complexes that mediate this process. The photosystem of GSB includes a large antenna complex called a chlorosome. After light is captured by the chlorosome, the photon energy is transferred through two pigment-binding proteins, the baseplate protein and the Fenna-Matthews-Olson or FMO protein, to the reaction center where excitation energy is converted to chemical energy. The membrane-attached FMO protein functions as a "wire" to transfer the excitation energy from the peripheral antenna chlorosome to the reaction center. The isolated FMO protein has long been a model system to understand energy transfer mechanisms and has been investigated by a large variety of spectroscopic and theoretical studies. In the thesis, the structural and functional properties of the FMO protein were further investigated by studying the protein isolated from different species and also a genetically modified version. In addition, the interaction network in vivo centered on the FMO protein was elucidated. The structure of the FMO protein from P. aestuarii 2K was solved to 1.3 A resolutions, and an 8th pigment was discovered. The nature and stoichiometry of the 8th pigment in the protein was studied by native electrospray mass spectrometry (MS) coupled to HPLC pigment analysis. The structure of the FMO protein from P. phaeum was also determined. The first FMO mutant generated by replacing the phytyl tail of the BChl a to geranylgeranyl in Chlorobaculum tepidum was characterized. Spectral and structural insights into the FMO protein were further gained from the comparative study of the FMO protein purified from a newly discovered sixth group of photosynthetic bacteria called Candidatus Chloracidobacterium thermophilum. The collection and study of the various FMO proteins have deepened our understanding of this antenna complex. The orientation of the FMO protein on the cytoplasmic membrane in vivo was determined by combining a specific chemical labeling method with MS analysis. The results gave the first experimental evidence that the BChl a #3 side of the protein is in close contact with the membrane. This MS-based specific protein surface mapping method was further developed to study protein-ligand interactions. Furthermore, the detailed interaction between the FMO protein and the chlorosome, specifically the baseplate protein at the bottom of the chlorosome, was investigated using hydrogen/deuterium exchange coupled with MS analysis. The high excitation energy transfer efficiency observed in photosynthetic organisms relies on the optimal pigment-protein binding geometry in the individual protein complexes and also on the overall architecture of the photosystems. On the basis of this work, a general picture of the photosystem from GSB can be constructed. Keywords: FMO protein; Energy transfer; Green sulfur bacteria; Native spray mass spectrometry; Protein surface mapping; Renewable energy