Studying functional properties of a cytochrome b6/f-Photosystem I supercomplex

Basic data for this project

Description

The function and performance of photosystem I (PSI) complexes are modulated by reversible association of light-harvesting proteins, the cytochrome b6f complex, or various redox components to form a mega-complex (CEF supercomplex) under varying environmental conditions. This remodeling of the PSI complex structure is an important regulatory strategy for photosynthetic organisms to cope with adverse environments. CEF-supercomplexes have only recently been isolated from C. reinhardtii and their functional properties have not been elucidated in detail. Successful isolation of a functional CEF supercomplex permits studying electron transfer within a cytochrome b6/f and PSI complex for the first time. It is our aim to elucidate CEF in isolated CEF supercomplex particles stemming from wild type, ANR1, CAS and FNR knockdown as well as in PGR5 and PGRL1 knockout mutants. Strikingly, FNR bound to the CEF-supercomplex can be photo-reduced by Fdx1 but not by Fdx2. Fdx2 has been shown to interact with ANR1. Thus, we aim to elucidate whether Fdx2 promotes CEF in vivo and in vitro. In the absence of PGR5 and PGRL1, the CEF-supercomplex is not formed, opening the option to investigate regulatory requirements for CEF-supercomplex assembly. For this purpose, pgrl1 and pgr5 will be genetically engineered and transformed into the double as well as into single mutants to measure supercomplex assembly and CEF function in vivo and in vitro, likely providing a link between regulation of CEF supercomplex assembly and CEF function. Besides we will elucidate the protein complex composition by quantitative mass spectrometry and chemical crosslinking approaches. In summary, these experiments will give us novel mechanistic insights into in vitro and in vivo functional properties of the CEF supercomplex.