Yukiteru Katsube

Tertiary structure of Bacillus thermoproteolyticus [4Fe-4S] ferredoxin: Evolutionary implications for bacterial ferredoxins

The structure of a low-potential [4Fe-4S] ferredoxin from Bacillus thermoproteolyticus has been solved using anomalous scattering data from iron atoms in the diffraction data of native crystals and refined partially to a crystallographic R-factor of 0.33, with 2.3 A (1 A = 0.1 nm) resolution data. The least-squares refinement based on the Bijvoet differences has determined that the four iron atoms in the cluster are an equal distance, approximately 2.8 A, apart. The NH ... S hydrogen bonds between polypeptide nitrogen atoms, and both cysteine and inorganic sulfur atoms, are present, as in ferrodoxin from Peptococcus aerogenes. The polypeptide chain of the B. thermoproteolyticus ferredoxin has a fold closely similar to that of 2[4Fe-4S] ferredoxin from P. aerogenes. The structural correspondence indicates strongly that both types of ferredoxin evolved from a common ancestor. The second cluster-binding region in P. aerogenes ferredoxin corresponds to the alpha-helix in B. thermoproteolyticus ferredoxin. The secondary-structure predictions strongly suggest that the alpha-helix is generally present in the monocluster-type ferredoxins. The conformational change to alpha-helix, insertions of a loop and a protrusion, as well as the absence of the second cluster in B. thermoproteolyticus ferredoxin, result in the lack of 2-fold symmetry present in P. aerogenes ferredoxin. So, the track of gene duplication is no longer detectable in the tertiary structure alone. The evolutionary events that may have occurred in the ferredoxins with the [4Fe-4S] cluster are discussed.

Structure of [4Fe-4S] ferredoxin from Bacillus thermoproteolyticus refined at 2.3 Å resolution: Structural comparisons of bacterial ferredoxins

The structure of a low-potential ferredoxin isolated from Bacillus thermoproteolyticus has been refined by a restrained least-squares method. The final crystallographic R factor is 0.204 for 2906 reflections with F greater than 3 sigma F in the 6.0 to 2.3 A resolution range. The model contains 81 amino acid residues, one [4Fe-4S] cluster, and 59 water molecules. The root-mean-square deviation from ideal values for bond lengths is 0.018 A, and the mean coordinate error is estimated to be 0.25 A. The present ferredoxin is similar in the topology of the polypeptide backbone to the dicluster-type ferredoxins from Peptococcus aerogenes and Azotobacter vinelandii, but has considerable insertions and deletions of the peptide segments as well as different secondary structures. Although all but the C-terminal C zeta atoms of P. aerogenes ferredoxin superpose on the C alpha atoms of A. vinelandii ferredoxin, only 60% superpose on the C alpha atoms of B. thermoproteolyticus ferredoxin, with a root-mean-square distance of 0.82 A for each pair. The conformations of the peptide segments surrounding the [4Fe-4S] clusters in these three ferredoxins are all conserved. Moreover, the schemes for the NH...S hydrogen bonds in these ferredoxins are nearly identical. The site of the aromatic ring of Tyr27 in B. thermoproteolyticus ferredoxin is close spatially to that of Tyr28 in P. aerogenes ferredoxin with reference to the cluster, but these residues do not correspond in the spatial alignment of their polypeptide backbones. We infer that in monocluster-type ferredoxins, the side-chain at the 27th residue has a crucial effect on the stability of the cluster. Of the four cysteine residues that bind to the second Fe-S cluster in the dicluster-type ferredoxins, two are conserved in the monocluster-type ferredoxins from Desulfovibrio gigas. D. desulfuricans Norway, and Clostridium thermoaceticum. The tertiary structure of B. thermoproteolyticus ferredoxin suggests that in such monocluster-type ferredoxins these two cysteine residues, which in it correspond to Ala21 and Asp53, form a disulfide bridge.

Structure-function relationship of [2Fe2S] ferredoxins and design of a model molecule

[2Fe-2S] ferredoxins isolated from various plants and algae comprise 93-99 amino acid residues and resemble each other not only in sequences, but also in physiological functions. One of them isolated from Spirulina platensis was subjected to X-ray analysis and its three dimensional structure is now known. [2Fe-2S] ferredoxins of a different type are found in halobacteria and comprise 128 amino acid residues. Both types of the [2Fe-2S] ferredoxins exhibit low redox potentials. By comparing the amino acid sequences of 28 [2Fe-2S] ferredoxins and the tertiary structure of S. platensis ferredoxin we predicted a common three-dimensional structure to the [2Fe-2S] ferredoxins and proposed a molecular surface area to be interacting with FNR. An artificial small molecule composed of 20 amino acid residues is designed on the basis of the tertiary structure of S. platensis ferredoxin. The amino acid sequence was predicted to be Pro-Tyr-Ser-Cys-Arg-Ala-Gly-Ala-Cys-Ser-Thr-Cys-Ala-Gyl-Pro-Leu-Leu-Thr Cys-Val which should have a [2Fe-2S] cluster with a low redox potential.

Preliminary X-ray diffraction studies on a [4Fe4S] ferredoxin from Bacillus thermoproteolyticus

Abstract A [4Feue5f84S] ferredoxin from Bacillus thermoproteolyticus has been crystallized. The space group is P1 with two molecules in the unit cell, with the dimensions a = 32.96 A , b = 37.83 A , c = 39.82 A , α = 118.1 °, β = 104.2 ° and γ = 89.7 ° . The Bijvoet-difference Patterson map of the native crystal shows up a prominent peak of [4Feue5f84S] cluster.

Intermolecular photoaddition involving 1,4-transfer of cyano group. 2. photoaddition of 6-cyano-1,3-dimethyluracil with acetylenic compounds

Abstract Wavelength dependent photoadditions of 6-cyano-1,3-dimethyluracil to phenylacetylenes are described. Photolysis with Prex-filtered light gave the fused cyclobutenes ( 3 , 9 ), whereas irradiation with 254-nm light afforded the rearranged adducts ( 5 , 11 ). The structures of the adducts were established by X-ray analyses.