In planta mutagenesis of Src homology 3 domain-like fold of NdhS, a ferredoxin-binding subunit of the chloroplast NADH dehydrogenase-like complex in Arabidopsis: a conserved Arg-193 plays a critical role in ferredoxin binding.
Chloroplast NADH dehydrogenase-like (NDH) complex mediates cyclic electron transport around photosystem I and chlororespiration in angiosperms. The Src homology 3 domain (SH3)-like fold protein NdhS/CRR31 is an NDH subunit that is necessary for high affinity binding of ferredoxin, indicating that chloroplast NDH functions as a ferredoxin:plastoquinone oxidoreductase. However, the mechanism of the interaction between NdhS and ferredoxin is unclear. In this study, we analyzed their interaction in planta by using site-directed mutagenesis of NdhS. In general, binding of ferredoxin to its target proteins depends on electrostatic interaction. In silico analysis predicted the presence of a positively charged pocket in the SH3-like domain of NdhS, where nine charged residues are highly conserved among plants. Systematic alteration of these sites with neutral glutamine revealed that only arginine 193 was required for high NDH activity in vivo. Further replacement of arginine 193 with negatively charged aspartate or glutamate or hydrophobic alanine significantly decreased the efficiency of ferredoxin-dependent plastoquinone reduction by NDH in ruptured chloroplasts. Similar results were obtained in in vivo analyses of NDH activity and electron transport. From these results, we propose that the positive charge of arginine 193 in the SH3-like domain of NdhS is critical for electrostatic interaction with ferredoxin in vivo.
From the Department of Botany, Graduate School of Science, Kyoto University, Kyoto 606-8502 and.
Hiroshi
H
Yamamoto
Toshiharu
T
Shikanai
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D017360:Arabidopsis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002736:Chloroplasts / Q000378:metabolism; D017124:Conserved Sequence; D005288:Ferredoxins / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009245:NADH Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases; D010971:Plastoquinone / Q000378:metabolism; D011485:Protein Binding; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055672:Static Electricity
D029681:Arabidopsis Proteins; D002352:Carrier Proteins; D005288:Ferredoxins; D021122:Protein Subunits; D009247:NADH, NADPH Oxidoreductases; C586745:NdhS protein, Arabidopsis; D009245:NADH Dehydrogenase; D010971:Plastoquinone
Arabidopsis; Chloroplast; Electron Transport; Photosynthesis; Protein-Protein Interactions