Purification and biochemical analysis of Physcomitrium patens flavodiiron protein heterocomplex

Oxygenic photosynthesis represents a key biochemical reaction by which plants, algae, and cyanobacteria convert light energy into biochemical energy. The complex photosynthetic apparatus needs to be finely regulated to properly perform the reaction according to the multiple environmental conditions that plants can experience. The photosynthetic electron transport chain is responsible for the light-dependent phase of photosynthesis, able to harvest light energy and produce NADPH and ATP. This complex machinery involves various regulatory mechanisms to protect itself, especially when exposed to light stress. Among these mechanisms, pseudo-cyclic electron flow (PCEF) mediated by flavodiiron proteins (FLV) represents an important safety valve for electrons in many photosynthetic organisms, including bryophytes. FLV accept electrons from Photosystem I (PSI) to catalyze oxygen reduction into water, creating a so-called water-water cycle that avoids electron accumulation at the level of PSI and prevents PSI photoinhibition under fluctuating light intensity. FLV are composed by three different functional domains. At N-terminal there is a metallo-β-lactamase-like domain harboring a diiron center followed by a flavodoxin-like domain containing FMN, while at C-terminal there is a NAD(P)H:flavin oxidoreductase-like domain able to interact with NADPH. Structural insights were partially obtained with prokaryotic FLV proteins, while only predicted structures are available for plant FLV proteins. This thesis deepened our knowledge about the biochemistry and the function of FLV proteins of Physcomitrium patens. This organism presents two FLV isoforms, FLVA and FLVB, which are thought to interact in the construction of a protein heterocomplex. Through the co-purification of the two isoforms, the occurrence of a strong FLVA-FLVB interaction was proven, using both Escherichia coli and Physcomitrium patens as expression systems. Furthermore, the heterocomplex was proven to bind both FMN and iron, which are crucial cofactors for FLV activity; and its enzymatic activity was deepened demonstrating the complex ability to accept electrons from NADH and perform oxygen reduction. Additionally, insights concerning the complex formation were obtained, revealing the important role of disulfide bridges in complex formation, and suggesting that a dimeric or even bigger association occurs. Altogether, these results constitute important achievements in FLV research and add knowledge to the understanding of photosynthetic machinery.