Two-dimensional pulsed electron spin resonance characterization of 15N-labeled archaeal Rieske-type ferredoxin

AbstractTwo-dimensional electron spin-echo envelope modulation (ESEEM) analysis of the uniformly 15N-labeled archaeal Rieske-type [2Fe–2S] ferredoxin (ARF) from Sulfolobus solfataricus P1 has been conducted in comparison with the previously characterized high-potential protein homologs. Major differences among these proteins were found in the hyperfine sublevel correlation (HYSCORE) lineshapes and intensities of the signals in the (++) quadrant, which are contributed from weakly coupled (non-coordinated) peptide nitrogens near the reduced clusters. They are less pronounced in the HYSCORE spectra of ARF than those of the high-potential protein homologs, and may account for the tuning of Rieske-type clusters in various redox systems

Structure and Molecular Evolution of CDGSH Iron-Sulfur Domains

The recently discovered CDGSH iron-sulfur domains (CISDs) are classified into seven major types with a wide distribution throughout the three domains of life. The type 1 protein mitoNEET has been shown to fold into a dimer with the signature CDGSH motif binding to a [2Fe-2S] cluster. However, the structures of all other types of CISDs were unknown. Here we report the crystal structures of type 3, 4, and 6 CISDs determined at 1.5 Å, 1.8 Å and 1.15 Å resolution, respectively. The type 3 and 4 CISD each contain one CDGSH motif and adopt a dimeric structure. Although similar to each other, the two structures have permutated topologies, and both are distinct from the type 1 structure. The type 6 CISD contains tandem CDGSH motifs and adopts a monomeric structure with an internal pseudo dyad symmetry. All currently known CISD structures share dual iron-sulfur binding modules and a β-sandwich for either intermolecular or intramolecular dimerization. The iron-sulfur binding module, the β-strand N-terminal to the module and a proline motif are conserved among different type structures, but the dimerization module and the interface and orientation between the two iron-sulfur binding modules are divergent. Sequence analysis further shows resemblance between CISD types 4 and 7 and between 1 and 2. Our findings suggest that all CISDs share common ancestry and diverged into three primary folds with a characteristic phylogenetic distribution: a eukaryote-specific fold adopted by types 1 and 2 proteins, a prokaryote-specific fold adopted by types 3, 4 and 7 proteins, and a tandem-motif fold adopted by types 5 and 6 proteins. Our comprehensive structural, sequential and phylogenetic analysis provides significant insight into the assembly principles and evolutionary relationship of CISDs

A 13-Kb Resolution Cosmid Map of the 14-Mb Fission Yeast Genome by Nonrandom Sequence-Tagged Site Mapping

We present the application of a nonrandom sequence-tagged site (STS) content detection method in mapping an entire genome, that of fission yeast. The novelty of our strategy is in the use of STS probes made from both ends of cosmid clones, selected on the basis of ''sample without replacement'' (only library clones that show no previous positive hybridization are selected and made into probes). We developed powerful techniques, based on consistency analysis, for error detection and contig assembly. In addition, we probed our library with genetically mapped markers and NotI or SfiI linking clones, thereby anchoring contigs onto chromosomes. Our map contains more than 1000 sites, including genes (most were previously unmapped), occurrences of known repetitive elements, and NotI-SfiI restriction sites

Resonance Raman characterization of archaeal and bacterial Rieske protein variants with modified hydrogen bond network around the [2Fe-2S] center

The rate of quinol oxidation by cytochrome bc1/b6f complex is in part associated with the redox potential (Em) of its Rieske [2Fe-2S] center, for which an approximate correlation with the number of hydrogen bonds to the cluster has been proposed. Here we report comparative resonance Raman (RR) characterization of bacterial and archaeal high-potential Rieske proteins and their site-directed variants with a modified hydrogen bond network around the cluster. Major differences among their RR spectra appear to be associated in part with the presence or absence of Tyr-156 (in the Rhodobacter sphaeroides numbering) near one of the Cys ligands to the cluster. Elimination of the hydrogen bond between the terminal cysteinyl sulfur ligand (St) and Tyr-Oη (as with the Y156W variant, which has a modified histidine Nɛ pKa,ox) induces a small structural bias of the geometry of the cluster and the surrounding protein in the normal coordinate system, and significantly affects some Fe-Sb/t stretching vibrations. This is not observed in the case of the hydrogen bond between the bridging sulfide ligand (Sb) and Ser-Oγ, which is weak and/or unfavorably oriented for extensive coupling with the Fe-Sb/t stretching vibrations

Mutation of the His ligand in mitoNEET stabilizes the 2Fe–2S cluster despite conformational heterogeneity in the ligand environment

The spectroscopic and stability properties and X-ray crystal structure of the H87C mutant of the 2Fe–2S ligand mitoNEET are reported. Strikingly, the single point mutation leads to changes in its absorbance and CD spectra and an increase of around sixfold in the stability of the 2Fe–2S clusters over the pH range 5–7. However, the crystal structure of the H87C mutant displays heterogeneity in a few key residues, including the introduced cysteine ligand. Nonetheless, the cluster is highly stabilized from release