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

Aimee M Terauchi,Shu-Fen Lu,Mirko Zaffagnini,Shane Tappa,Masakazu Hirasawa,Jatindra N Tripathy,David B Knaff,Patrick J Farmer,Stéphane D Lemaire,Toshiharu Hase,Sabeeha S Merchant

doi:10.1074/jbc.m109.023622

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 [Fe2S2] 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 (Em = -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.

Highlights

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 [1,2,3]
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
The most well known Fd-dependent reaction is the transfer of electrons from photosystem I (PSI) to NADPH, catalyzed by Fd:NADPϩ oxidoreductase (FNR)

Summary

The abbreviations used are

Ferredoxin; PSI, photosystem I; FNR, Fd:NADPϩ oxidoreductase; NiR, nitrite reductase; TRX, thioredoxin; FTR, Fd:thioredoxin reductase; TAP, Tris acetate-phosphate; MOPS, 4-morpholinepropanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. There is one non-photosynthetic ferredoxin located in the roots, AtFd3, which is nitrate-inducible This protein has higher electron transfer activity with sulfite reductase in in vitro assays compared with other Arabidopsis ferredoxin isoforms, suggesting in vivo function of AtFd3 in nitrate and sulfate assimilation [15, 17]. We hypothesize that the presence of as many as six ferredoxin isoforms in a single-celled organism like C. reinhardtii allows for the differential regulation of each isoform and the prioritization of reducing power toward certain metabolic pathways under changing environmental conditions To test this hypothesis, expression of the genes (PETF and FDX2– FDX6) encoding the six ferredoxin isoforms in Chlamydomonas reinhardtii was monitored under various conditions in which well characterized ferredoxin-dependent enzymes are known to be expressed. In the case of FDX2 whose product is most similar to classical Fd, we suggest that it has specificity for nitrite reductase based on its pattern of expression and activity with nitrite reductase

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION