Identification of global ferredoxin interaction networks in Chlamydomonas reinhardtii

Ferredoxins (FDXs) can distribute electrons originating from photosynthetic water oxidation, fermentation, and other reductant-generating pathways to specific redox enzymes in different organisms. The six FDXs identified in Chlamydomonas reinhardtii are not fully characterized in terms of their biological function. In this report, we present data from the following: (a) yeast two-hybrid screens, identifying interaction partners for each Chlamydomonas FDX; (b) pairwise yeast two-hybrid assays measuring FDX interactions with proteins from selected biochemical pathways; (c) affinity pulldown assays that, in some cases, confirm and even expand the interaction network for FDX1 and FDX2; and (d) in vitro NADP+ reduction and H2 photo-production assays mediated by each FDX that verify their role in these two pathways. Our results demonstrate new potential roles for FDX1 in redox metabolism and carbohydrate and fatty acid biosynthesis, for FDX2 in anaerobic metabolism, and possibly in state transition. Our data also suggest that FDX3 is involved in nitrogen assimilation, FDX4 in glycolysis and response to reactive oxygen species, and FDX5 in hydrogenase maturation. Finally, we provide experimental evidence that FDX1 serves as the primary electron donor to two important biological pathways, NADPH and H2 photo-production, whereas FDX2 is capable of driving these reactions at less than half the rate observed for FDX1.