X-ray Crystallographic and Solution State Nuclear Magnetic Resonance Spectroscopic Investigations of NADP⁺ Binding to Ferredoxin NADP Reductase from Pseudomonas aeruginosa^,

Abstract

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.

Structural Characterization of the Hemophore HasAp from Pseudomonas aeruginosa: NMR Spectroscopy Reveals Protein−Protein Interactions between Holo-HasAp and Hemoglobin,

“…It is likely that cross-peaks corresponding to Ser2 and Ser103 cannot be detected due to conformational disorder because Ser2 becomes the amino terminal residue upon cleavage of the initiator Met during expression in E. coli and Ser 103 is located in the loop exhibiting the highest B-factors in the X-ray crystal structure. Resonances originating from several of the remaining residues listed in Table 2 were assigned with the aid of samples selectively labeled with 15 N-Val, 15 N-Thr, 15 N-Leu, or 15 N-Tyr and 2D 1 H- 15 N HSQC experiments tailored to detect fast relaxing signals (37), in a manner similar to that used for the assignment of fast relaxing signals in heme oxygenase (18) and FPR (38) from Pseudomonas aeruginosa . For example, the HSQC spectrum of 15 N-Val HasAp acquired with standard conditions (Figure 6-A) displays 8 cross-peaks corresponding to previously assigned Val residues.…”

Structural, NMR Spectroscopic, and Computational Investigation of Hemin Loading in the Hemophore HasAp from Pseudomonas aeruginosa

“…Samples selectively labeled with 15 N-Gly, 15 N-Ala, or 15 N-Val were prepared with the above-described protocols supplementing the M9 media according to previously reported procedures 21–23…”

Bacterioferritin: Structure Function and Protein–Protein Interactions

“…104 Nuclear magnetic resonance (NMR) spectroscopic and X-ray crystallographic studies conducted with Pa FPR showed, for the first time, the interactions between a bacterial FPR and NADP + ; the cofactor binds to a preformed binding site on the structure of Pa FPR and the complex is stabilized by interactions between the protein and the 2′P AMP portion of the cofactor. 107 Prior to the structural characterization of the Ftn-like molecules in P. aeruginosa, 64,67 it was thought that the bacterium harbored a Bfr assembled from two distinct types of subunits, termed α and β 108 Studies carried out in 1999 demonstrated the presence of two genes encoding Ftn-like molecules in P. aeruginosa, which were named bacterioferritin A (bfrA) and bfrB. 109 The authors also showed that P. aeruginosa mutants lacking the bfrA gene express 50% of the catalase activity of the wild-type cell but that full catalase activity could be rescued by complementation with a plasmid expressing the bfrA gene.…”