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

Abstract

The crystal structure of the ISC-like [2Fe-2S] ferredoxin (FdxB), probably involved in the de novo iron-sulfur cluster biosynthesis (ISC) system of Pseudomonas putida JCM 20004, was determined at 1.90-Å resolution and displayed a novel tail-to-tail dimeric form. P. putida FdxB lacks the consensus free cysteine usually present near the cluster of ISC-like ferredoxins, indicating its primarily electron transfer role in the iron-sulfur cluster. Orientation-selective electron-nuclear double resonance spectroscopic analysis of reduced FdxB in conjunction with the crystal structure has identified the innermost Fe2 site with a high positive spin population as the nonreducible iron retaining the Fe(3+) valence and the outermost Fe1 site as the reduced iron with a low negative spin density. The average g (max) direction is skewed, forming an angle of about 27.3° (±4°) with the normal of the [2Fe-2S] plane, whereas the g (int) and g (min) directions are distributed in the cluster plane, presumably tilted by the same angle with respect to this plane. These results are related to those for other [2Fe-2S] proteins in different electron transport chains (e.g. adrenodoxin) and 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.

A rapid and robust method for selective isotope labeling of proteins

“…The fdxB gene coding for the ISC-like [2Fe-2S] ferredoxin (FdxB) of P. putida JCM 20004 (formerly P. ovalis IAM 1002) has been cloned and sequenced as a part of its isc gene cluster (DDBJ/EMBL/GenBank code AB109467) and heterologously overexpressed in E. coli BL21-CodonPlus(DE3)-RIL strain (Stratagene) using a pET28aFDXB-SG vector (based on a pET28a His-tag expression vector, Novagen), purified, and crystallized[19]. The structure of the recombinant FdxB has been determined and refined at 1.90-Å resolution (the final R -factor, 0.182; free- R factor, 0.216), and the coordinates and structural factors have been deposited in the Protein Data Bank (PDB ID code: 3AH7)[19].…”

“…The structure of the recombinant FdxB has been determined and refined at 1.90-Å resolution (the final R -factor, 0.182; free- R factor, 0.216), and the coordinates and structural factors have been deposited in the Protein Data Bank (PDB ID code: 3AH7)[19]. …”

“…This system was suitable for heterologous overproduction of the uniformly 15 N-labeled iron-sulfur holoproteins by employing the combination of a pET28a vector (Novagen) plus E. coli CodonPlus(DE3)-RIL host strain (Stratagene) system, because our previous overproduction procedures using the combinations of the pTrc99A vector/ E. coli CodonPlus(DE3)-RIL host strain/M9 salt-based synthetic medium system[28,29] were much less successful with the P. putida fdxB gene, giving negligible amount of a recombinant holo-protein (insufficient for high-resolution pulsed EPR analysis of iron-sulfur proteins typically requiring ~0.4 mM or more), for reasons that are not clear. The cells (from 1L culture medium in 2L-flask) were pelleted by centrifugation, and the U-FdxB holoprotein was purified as reported previously for the nonlabeled protein[19]. The purified protein was concentrated with Centriprep-10 and Microcon-YM10 apparatus (Amicon) to ~3 mM, rapidly frozen in liquid nitrogen, and stored at −80 °C until use.…”

“…Using the newly engineered C43(DE3) auxotrophs, we demonstrate their utility using two proteins: 1) the cytochrome bo 3 (cyt bo 3 ) ubiquinol oxidase[18] (CyoABCD) from E. coli , a 144 kDa membrane protein containing 4 transmembrane subunits, two heme prosthetic groups, one ubiquinone and one copper; 2) a small (13 kDa), water-soluble [2Fe-2S] ferredoxin[19] (FdxB) from Pseudomonas putida thought to be involved in the assembly of Fe-S clusters. Three cyt bo 3 samples were prepared, in which Ile/Leu, Ile/Tyr and His/Tyr amino acid pairs were, respectively, enriched with 13 C and 15 N. The MAS SSNMR experiments performed with these samples indicated clean and nearly complete 13 C and 15 N isotopic enrichment of the selected amino acid types.…”

The competition between chemistry and biology in assembling iron–sulfur derivatives. Molecular structures and electrochemistry. Part II. {[Fe2S2](SγCys)4} proteins

“…putida JCM 20004 (previously known as Pseudomonas ovalis) is a non-pathologic variant of P. putida [70]. In the solid state it is an homodimer containing two [2Fe-2S] ferredoxins [71], but no data are available about the redox potentials of such centers.…”