The solution structure of parsley [2Fe‐2S]ferredoxin

Abstract

The [2Fe-2S]ferredoxin I (Fd I) from parsley leaves (M r ϭ 10500; 96 amino acids) in the Fe(III)-Fe(III) oxidized form has been studied by 1 H-NMR spectroscopy. Sequence-specific 1 H-NMR assignments were obtained through two-dimensional classical double-quantum-filtered-COSY, NOESY and TOCSY spectra. NOEs between protons as close as 5.6 Å from the paramagnetic Fe(III) atoms were observed at 800 MHz. A total of 3066 NOEs (of which 2533 are meaningful) and 18 distance constraints taken from X-ray crystallography of the Fe 2 S 2 active site were used to obtain the solution structure. From inversion recovery NOESY experiments, 33 longitudinal relaxation rate (AE para ) constraints were used for the structural refinement. The final structure was obtained by a process of restrained energy minimization. Root-mean-square (rmsd) deviation values obtained for the family of 18 structures (with reference to the average structure) are 0.52 Ϯ0.10 Å and 0.91Ϯ 0.12 Å for backbone and all heavy atoms respectively. The structure consists of seven-strands of β-sheets and four short A-helices. The quality of the present solution structure is among the best of those reported for [2Fe-2S]ferredoxins. The secondary structure and overall folding are compared with those of Anabaena variabilis Fd and the higher plant Equistum arvense (horse tail) protein determined through X-ray crystallography. The groups believed to be responsible for electron transfer have been analysed.Keywords : ferredoxin; solution structure; paramagnetism. Plant [2Fe-2S]ferredoxins are very important redox components involved in photosystem I (PSI) electron transport [1Ϫ3].They perform one-electron transfer (ET) from subunit PsaC of PSI to ferredoxin NADP-reductase (FNR), ferredoxin nitrite oxidoreductase, glutamate synthase and sulfite reductase [4]. There are now more than 75 amino acid sequences for this class of protein from higher plants, algae and cyanobacteria [1] (Table 1). Plant ferredoxins are very acidic (pI ϭ 3.4), with a charge balance of Ϫ18(Ϯ 2) at pH 7.5 from amino acid compositions. From X-ray crystallography on cyanobacteria PSI (6 Å resolution), it appears that the PSI subunits PsaD, PsaE and PsaC are involved in establishing an electrostatic orientation of the located on Fe(I), which is the Fe closest to the protein surface (Ϸ5 Å ) [17].The NMR technique is a powerful method for determining solution structures of small biomolecules. At first it was believed that it would be very difficult (or impossible) to detect a sufficiently large number of constraints for solution structure determination of paramagnetic metalloproteins. Since the first NMR solution structure of a paramagnetic protein in 1994 [18], new strategies and approaches have been introduced. In the few NMR solution structures of oxidized [2Fe-2S]ferredoxins reported from Pseudomonas putida [19], Synechocystis sp. PCC 6803 [20] and Synechococcus elongatus [21], the quality of the structures is severely hampered by the lack of NOEs closer than about 8 Å from the iron atoms, as previou...

Water-assisted Proton Transfer in Ferredoxin I

“…Furthermore, the experimental rate k on hop ϭ 1294 s Ϫ1 was measured at a pH of 8.34. At a physiologically more relevant acidic pH (69,70), the experimental rate for PT2 is smaller (k off hop ϭ 36 Ϯ 5 s Ϫ1 at pH 5.0 (2)), which, according to transition state theory, corresponds to a barrier increase by ⌬G PT2 ‡ ϭ 1.4 kcal/mol compared with pH 8.34. In the simulations for PT2, the carboxyl group of Asp 15 is deprotonated, which covers the situation down to a pH of Ϸ 4.1 (the pK a of Asp lies at 4.1 (71)).…”

Discrimination between Distant Homologs and Structural Analogs: Lessons from Manually Constructed, Reliable Data Sets

“…47,48 2Fe-2S ferredoxins (also referred to as β-grasp ferredoxins) are electron transporters in photosynthesis and nitrogen fixation. 49,50 TGS domain is named after three proteins [ThrRS, guanosine 5′-triphosphatase (GTPase), and SpoT] in which it is found, 51 and the TGS-like superfamily is represented by the N1 domain of the threonyl-tRNA synthetase 52 that may participate in the proofreading activity of this enzyme. 53 CAD and PB1 domains mediate protein complex formation through heterodimerization.…”

Solution structure of HndAc: A thioredoxin‐like domain involved in the NADP‐reducing hydrogenase complex

“…As a consequence, the amide resonances of residues C12–F18 and R52–I62 are missing in the HSQC spectrum. In order to circumvent this difficulty, an accepted approach is to model the missing part based on structural homology (Lelong et al 1995; Baumann et al 1996; Im et al 1998; Mo et al 1999; Pochapsky et al 1999; Müller et al 2002). The backbone dihedral angle constraints from the X‐ray structure of the thioredoxin from A. aeolicus (PDB code 1F37) were selected as a template to model the residues V11–A22 and D49–V65 of HndAc (Fig.…”