Pattern of Expression and Substrate Specificity of Chloroplast Ferredoxins from Chlamydomonas reinhardtii

Abstract

Ferredoxin (Fd) is the major iron-containing protein in photosynthetic organisms and is central to reductive metabolism in the chloroplast. The Chlamydomonas reinhardtii genome encodes six plant type [Fe 2 S 2 ] ferredoxins, products of PETF, FDX2-FDX6. We performed the functional analysis of these ferredoxins by localizing Fd, Fdx2, Fdx3, and Fdx6 to the chloroplast by using isoform-specific antibodies and monitoring the pattern of gene expression by iron and copper nutrition, nitrogen source, and hydrogen peroxide stress. In addition, we also measured the midpoint redox potentials of Fd and Fdx2 and determined the kinetic parameters of their reactions with several ferredoxin-interacting proteins, namely nitrite reductase, Fd:NADP ؉ oxidoreductase, and Fd:thioredoxin reductase. We found that each of the FDX genes is differently regulated in response to changes in nutrient supply. Moreover, we show that Fdx2 (E m ‫؍‬ ؊321 mV), whose expression is regulated by nitrate, is a more efficient electron donor to nitrite reductase relative to Fd. Overall, the results suggest that each ferredoxin isoform has substrate specificity and that the presence of multiple ferredoxin isoforms allows for the allocation of reducing power to specific metabolic pathways in the chloroplast under various growth conditions.Ferredoxins are small (ϳ11,000-kDa), soluble, iron-sulfur cluster-containing proteins with strongly negative redox potentials (Ϫ350 to Ϫ450 mV) that function as electron donors at reductive steps in various metabolic pathways (1-3). In photosynthetic organisms, the well studied ferredoxin (Fd 4

Characterization of the Key Step for Light-driven Hydrogen Evolution in Green Algae

“…Furthermore, reduced Fdx2 obviously has a substrate specificity for nitrite reductase. This behavior suggests an equivalent role of Fdx2 and root-type ferredoxin of higher plants, which operates independently from the photosynthetic electron transport (38).…”

“…Besides PETF, the nuclear genome of C. reinhardtii contains at least five additional genes encoding proteins with significant homology to plant-type ferredoxins (FDX2-6) (37,38). Compared with PetF, Fdx2 exhibits the highest level of sequence homology (65% identity), although for the other ferredoxins the similarity lies between 47% (Fdx5) and 29% (Fdx6) sequence identity.…”

Identification of Global Ferredoxin Interaction Networks in Chlamydomonas reinhardtii

“…All six genes encode proteins with typical [2Fe-2S]-binding domains, containing the conserved cysteine residues required for [FeS] cluster ligation. Phylogenetic analysis indicates that FDX1 and FDX5 are most closely related to photosynthetic leaf-type FDXs, whereas FDX2 appears to be a nonphotosynthetic root-type FDX (16). FDX3, FDX4, and FDX6 phylogenetically cluster with FDXs of unknown function, as well as with cyanobacterial FDXs.…”

Critical role of Chlamydomonas reinhardtii ferredoxin-5 in maintaining membrane structure and dark metabolism

“…FDX1 also accepts electrons from FDX-NADP oxidoreductase (FNR) (7) and donates electrons to HYDA hydrogenases (5,8,9). Other FDXs may be involved in state transitions, nitrogen metabolism, cellular responses to reactive oxygen species (ROS), and dark anoxia (4,5,(10)(11)(12)(13).…”

“…The Chlamydomonas genome encodes 13 FDXs (SI Appendix, Table S1) with localization and redox properties that suggest involvement in specific redox reactions (4). Using a yeast two hybrid approach, a global FDX interaction network was established for Chlamydomonas, suggesting putative roles for FDX1 (originally designated Fd) in redox metabolism, carbohydrate modification, and fatty acid biosynthesis (5); this FDX was already known to function in both linear and cyclic photosynthetic electron flow (4,6). FDX1 also accepts electrons from FDX-NADP oxidoreductase (FNR) (7) and donates electrons to HYDA hydrogenases (5,8,9).…”