X-ray crystallographic and Solution State NMR Spectroscopic Investigations of NADP+ Binding to Ferredoxin-NADP Reductase (FPR) from Pseudomonas aeruginosa

摘要

The >ferredoxin nicotinamide adenine dinucleotide >phosphate >reductase from Pseudomonas aeruginosa (pa-FPR) in complex with NADP⁺ has been characterized by X-ray crystallography and in solution by NMR spectroscopy. The structure of the complex revealed that pa-FPR harbors a pre-formed NADP⁺ binding pocket where the cofactor binds with minimal structural perturbation of the enzyme. These findings were complemented by obtaining sequential backbone resonance assignments of this 29,518 kDa enzyme, which enabled the study of the pa-FPR-NADP complex by monitoring chemical shift perturbations induced by addition of NADP⁺ or the inhibitor, adenine dinucleotide phosphate (ADP), to pa-FPR. The results are consistent with a pre-formed NADP⁺ binding site and also demonstrate that the pa-FPR-NADP complex is largely stabilized by interactions between the protein and the 2′P AMP portion of the cofactor. Analysis of the crystal structure also shows a vast network of interactions between the two cofactors, FAD and NADP⁺, and the characteristic AFVEK²⁵⁸ C’-terminal extension that is typical of bacterial FPRs but is absent in their plastidic >ferredoxin >NADP⁺ reductase (FNR) counterparts. The conformations of NADP⁺ and FAD in pa-FPR place their respective nicotinamide and isoalloxazine rings 15 Å apart and separated by residues in the C’-terminal extension. The network of interactions between NADP⁺, FAD and residues in the C’-terminal extension indicate that the gross conformational rearrangement that would be necessary to place the nicotinamide and isoalloxazine rings parallel and adjacent to one another for direct hydride transfer between NADPH and FAD in pa-FPR is highly unlikely. This conclusion is supported by observations made in the NMR spectra of pa-FPR and pa-FPR-NADP, which strongly suggest that residues in the C’-terminal sequence do not undergo conformational exchange in the presence or in the absence of NADP⁺. These findings are discussed in the context of a possible stepwise electron-proton-electron transfer of hydride in the oxidation of NADPH by FPR enzymes.