Journal:JBIC:12

ISC-like [2Fe-2S] ferredoxin (FdxB) dimer from Pseudomonas putida JCM 20004: Structural and electron nuclear double resonance characterization

Toshio Iwasaki^[1]

Molecular Tour
Biological iron-sulfur (Fe-S) clusters are functionally versatile, modular prosthetic groups. The electronic structure and the site of iron reduction of these protein-bound cofactors account for the electron transfer function and mechanism. In the present work we have solved the structure of the ISC-like [2Fe-2S] ferredoxin called FdxB from the non-pathogenic gammaproteobacterium Pseudomonas putida JCM 20004 (formerly Pseudomonas ovalis IAM 1002). This FdxB protein contains an adrenodoxin (Adx) like, redox-active [2Fe-2S] cluster, which plays an essential role in the de novo iron-sulfur cluster assembly (ISC) system. It is encoded by the fdxB gene as a constituent of the cognate iscR-iscS1-iscU-iscA-hscB-hscA-fdxB gene cluster for the ISC system (DDBJ-EMBL-GenBank code AB109467). In P. putida the ISC pathway is apparently the sole system for in vivo Fe-S cluster assembly whereas the SUF pathway is missing in the bacterial genome (unlike in Escherichia coli).

The FdxB structure has a βαββαβ fold with the β-grasp/ubiquitin-like fold motif as found in regular eukaryal and bacterial [2Fe-2S] ferredoxins (e.g. 1i7h.pdb, 1cje.pdb, 1e9m.pdb). FdxB is folded into an (α+β) core fold domain and an extended C-terminal tail. In the lattice FdxB was found to be homo-dimeric, displaying the isologous association of the extended C-terminal tail from each protomer. Each protomer binds a [2Fe-2S] cluster that is coordinated by four terminal cysteine sulfur atoms, where the outermost iron (Fe1) near the protein surface is coordinated by Cys41Sγ and Cys47Sγ and the innermost iron (Fe2) by Cys50 Sγ and Cys86 Sγ. In the dimeric structure, two [2Fe-2S] clusters are separated at the closest iron-to-iron (Fe1-Fe1) distance of 25 ֵ, suggesting that a rapid interprotomer electron transfer between them would be unlikely to occur. In the place of the consensus free cysteine usually present near the [2Fe-2S] cluster of ISC-like ferredoxins, FdxB has the Lys45 side chain which forms a salt-bridge interaction with Asp65 Oδ2. Thus, the overall FdxB structural features argue for its primarily electron transfer role in the cognate ISC system, rather than the direct catalytic function.

With the molecular structural frame determined from the FdxB structure, our electron-nuclear double resonance (ENDOR) analysis has allowed to determine the average gmax direction of the reduced FdxB, which is skewed, pointing roughly towards Cys50 Cα and forming an angle of about 27.3 (±4) degrees with the normal of the [2Fe-2S] plane, while the gint- and gmin-directions are distributed in a plane tilted toward the cluster plane. The site of reduced iron in the reduced FdxB is the outermost Fe1 site with the low negative spin density, while the innermost Fe2 site with the high positive spin population is the non-reducible iron retaining the Fe3+-valence of a reduced cluster. From a structural point of view, the larger number of polarized (or polarizable) bonds (NH, OH) and the extended hydrogen bonding network around Fe1 in FdxB may be the crucial factor favoring the accommodation of the reducing electron at the outermost Fe1 site. These results suggest a significant distortion of the electronic structure of the reduced [2Fe-2S] cluster under the influence of the protein environment around each iron site in general.