1H AND 15N NMR Sequential Assignment, Secondary Structure, and Tertiary Fold of [2Fe-2S] Ferredoxin from Synechocystis sp. PCC 6803

Abstract

The [2Fe-2S] ferredoxin extracted from Synechocystis sp. PCC 6803 was studied by 1H and 15N nuclear magnetic resonance. Sequence-specific 1H and 15N assignment of amino acid residues far from the paramagnetic cluster (distance higher than 8 A) was performed. Interresidue NOE constraints have allowed the identification of several secondary structure elements: one beta sheet composed of four beta strands, one alpha helix, and two alpha helix turns. The analysis of interresidue NOEs suggests the existence of a disulfide bridge between the cysteine residues 18 and 85. Such a disulfide bridge has never been observed in plant-type ferredoxins. Structure modeling using the X-PLOR program was performed with or without assuming the existence of a disulfide bridge. As a result, two structure families were obtained with rms deviations of 2.2 A. Due to the lack of NOE connectivities resulting from the paramagnetic effect from the [2Fe-2S] cluster, the structures were not well resolved in the region surrounding the [2Fe-2S] cluster, at both extremities of the alpha helix and the C and N terminus segments. In contrast, when taken separately, the beta sheet and the alpha helix were well defined. This work is the first report of a structure model of a plant-type [2Fe-2S] Fd in solution.

Molecular analysis of the soluble butane monooxygenase from ‘Pseudomonas butanovora’ a aThe GenBank accession number for the bmoXYBZDC sequence is AY093933.

“…The sequence analysis and comparison (data not shown) reveals a [2Fe-2S] binding domain (ferredoxin domain) similar to those found in plants and bacteria (Lelong et al, 1995). The cysteine ligands to ferredoxin iron-sulfur centres are conserved in BMOR and located at residues 43, 48, 51 and 84 (42, 47, 50 and 82 in the methane monooxygenase reductase -MMOR -from Methylococcus capsulatus).…”

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

“…The distance between the two cysteine sulfurs of Synechocystis sp. PCC 6803 Fd in solution was reported to be shorter (Ϸ7.3 Å ) than that from the corresponding [2Fe-2S] X-ray structures (Ϸ10 Å ) [20]. The case of parsley Fd I is clearcut, because this protein has only one free cysteine (Cys18) and no disulfide bridge in that region can exist.…”

“…The bond lengths between Fe and S of different amino acids were given upper distance limits by using data taken as averages from X-ray crystallography. In previous investigations of [2Fe-2S] proteins, the modeling of the cluster was achieved by imposing 41 constraints from X-ray data [19,20]. Since the primary structure of the present protein is similar to that of proteins for which the X-ray structure is available (Table 1), we succeeded in solving the structure by taking from X-ray data only 18 distances between iron and sulfur atoms in the cluster (14 plus 4 additional covalent bonds in the two CysFe-S residues) without imposing any additional constraints to the surrounding amino acids.…”

“…Such a bridge could be responsible for the absence of the β-sheet region 82Ϫ83 [20]. However, the disulfide bridge has never been observed in X-ray crystallographic studies.…”

“…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 previously noted [22]. To achieve a reasonable folding it was necessary to introduce several bond distances and angles derived from the X-ray crystal structure for the [2Fe-2S] active centers as well as for nearby residues.…”