Inter-domain interaction of ferredoxin-NADP+ reductase important for the negative cooperativity by ferredoxin and NADP(H)

• Published in: Journal of biochemistry (Tokyo) 2023-09, Vol.174 (4), p.327-334
• Main Authors: Kimata-Ariga, Yoko, Shinkoda, Rina, Abe, Ryuya
• Format: Article
• Language: English
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/jb/mvad046

Ferredoxin-NADP+ reductase (FNR) in plants receives electrons from ferredoxin (Fd) and converts NADP+ to NADPH. The affinity between FNR and Fd is weakened by the allosteric binding of NADP(H) on FNR, which is considered as a part of negative cooperativity. We have been investigating the molecular mechanism of this phenomenon, and proposed that the NADP(H)-binding signal is transferred to the Fd-binding region across the two domains of FNR, NADP(H)-binding domain and FAD-binding domain. In this study, we analyzed the effect of altering the inter-domain interaction of FNR on the negative cooperativity. Four site-directed FNR mutants at the inter-domain region were prepared, and their NADPH-dependent changes in the Km for Fd and physical binding ability to Fd were investigated. Two mutants, in which an inter-domain hydrogen bond was changed to a disulfide bond (FNR D52C/S208C) and an inter-domain salt bridge was lost (FNR D104N), were shown to suppress the negative cooperativity by using kinetic analysis and Fd-affinity chromatography. These results showed that the inter-domain interaction of FNR is important for the negative cooperativity, suggesting that the allosteric NADP(H)-binding signal is transferred to Fd-binging region by conformational changes involving inter-domain interactions of FNR.