Nanomechanical Study of Enzyme: Coenzyme Complexes: Bipartite Sites in Plastidic Ferredoxin-NADP+ Reductase for the Interaction with NADP.

Sandra Pérez-Domínguez,Carlos Marcuello,Silvia Caballero-Mancebo,Marta Martínez-Júlvez,Milagros Medina,Anabel Lostao

doi:10.3390/antiox11030537

Abstract

Plastidic ferredoxin-NADP+ reductase (FNR) transfers two electrons from two ferredoxin or flavodoxin molecules to NADP+, generating NADPH. The forces holding the Anabaena FNR:NADP+ complex were analyzed by dynamic force spectroscopy, using WT FNR and three C-terminal Y303 variants, Y303S, Y303F, and Y303W. FNR was covalently immobilized on mica and NADP+ attached to AFM tips. Force–distance curves were collected for different loading rates and specific unbinding forces were analyzed under the Bell–Evans model to obtain the mechanostability parameters associated with the dissociation processes. The WT FNR:NADP+ complex presented a higher mechanical stability than that reported for the complexes with protein partners, corroborating the stronger affinity of FNR for NADP+. The Y303 mutation induced changes in the FNR:NADP+ interaction mechanical stability. NADP+ dissociated from WT and Y303W in a single event related to the release of the adenine moiety of the coenzyme. However, two events described the Y303S:NADP+ dissociation that was also a more durable complex due to the strong binding of the nicotinamide moiety of NADP+ to the catalytic site. Finally, Y303F shows intermediate behavior. Therefore, Y303, reported as crucial for achieving catalytically competent active site geometry, also regulates the concerted dissociation of the bipartite nucleotide moieties of the coenzyme.

Highlights

Close to the mechanical strength found for the WT AnFNRox :NADP+ complex, we find azurin:cytochrome c551 [46] (140 pN) and p53:Mdm2 (130 pN) complexes [47]
The presented data provide a comprehensive dissection of the ferredoxin-NADP+ reductase (FNR):NADP+ dissociation process
They lead to novel conclusions about the kinetics and energetics of the binding of the NADP+ substrate to FNR, and highlight the bipartite coenzyme binding/dissociation mode and the role of the enzyme C-terminal Tyr residue in the process

Summary

Introduction

Enzymatic processes are catalyzed by the cooperation of a number of enzymes and coenzymes that, in successive steps, transform metabolites into a variety of products. Enzymes are exploited in a variety of manufacturing processes such as the synthesis of medicines [1], food processing, purifying factory effluents, and pollution in water and soils [2], among others. Despite the great power of enzymatic processes, their potential has not been fully exploited, mainly due to the limited understanding of the mechanisms regarding the assembly and dissociation between reacting molecules. Recent advances in imaging methods have demonstrated that it is possible to make direct observations of the dynamic behavior of single molecules [3,4] and to determine the Antioxidants 2022, 11, 537.

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