Analysis of covalent flavinylation using thermostable succinate dehydrogenase from Thermus thermophilus and Sulfolobus tokodaii lacking SdhE homologs.

Recent studies have indicated that post-translational flavinylation of succinate dehydrogenase subunit A (SdhA) in eukaryotes and bacteria require the chaperone-like proteins Sdh5 and SdhE, respectively. How does covalent flavinylation occur in prokaryotes, which lack SdhE homologs? In this study, I showed that covalent flavinylation in two hyperthermophilic bacteria/archaea lacking SdhE, Thermus thermophilus and Sulfolobus tokodaii, requires heat and dicarboxylic acid. These thermophilic bacteria/archaea inhabit hot environments and are said to be genetically far removed from mesophilic bacteria which possess SdhE. Since mesophilic bacteria have been effective at covalent bonding in temperate environments, they may have caused the evolution of SdhE.

Crystallization and preliminary X-ray diffraction studies of hyperthermophilic archaeal Rieske-type ferredoxin (ARF) from Sulfolobus solfataricus P1.

The hyperthermophilic archaeal Rieske-type [2Fe-2S] ferredoxin (ARF) from Sulfolobus solfataricus P1 contains a low-potential Rieske-type [2Fe-2S] cluster that has served as a tractable model for ligand-substitution studies on this protein family. Recombinant ARF harbouring a pET30a vector-derived N-terminal extension region plus a hexahistidine tag has been heterologously overproduced in Escherichia coli, purified and crystallized by the hanging-drop vapour-diffusion method using 0.05 M sodium acetate, 0.05 M HEPES, 2 M ammonium sulfate pH 5.5. The crystals diffracted to 1.85 A resolution and belonged to the tetragonal space group P4(3)2(1)2, with unit-cell parameters a = 60.72, c = 83.31 A. The asymmetric unit contains one protein molecule.

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

Two-dimensional electron spin-echo envelope modulation (ESEEM) analysis of the uniformly (15)N-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.

Continuous-wave and pulsed EPR characterization of the [2Fe-2S](Cys)3(His)1 cluster in rat MitoNEET.

CW EPR spectra of reduced [2Fe-2S](Cys)(3)(His)(1) clusters of mammalian mitoNEET soluble domain appear to produce features resulting from the interaction of the electron spins of the two adjacent clusters, which can be explained by employing the local spin model. This model favors the reduction of the outermost iron with His87 and Cys83 ligands, which is supported by orientation-selected hyperfine sublevel correlation (HYSCORE) characterization of the uniformly (15)N-labeled mitoNEET showing one strongly coupled nitrogen from the His87 N(delta) ligand with hyperfine coupling (15)a = 8 MHz. The (14)N and (15)N HYSCORE spectra also exhibit at least two different cross-peaks located near diagonal in the (++) quadrant, with frequencies approximately 2.8 and 2.4 MHz (N2), and the other approximately 4.0 and 3.5 MHz (N1), but did not show any of the larger splitting approximately 1.1-1.4 MHz previously seen with Rieske proteins. Further analysis with partially (15)N(3)-His-labeled protein indicates that His87 N(epsilon) cross-peaks produce resolved features (N2) in the (14)N spectrum but contribute much less than weakly coupled peptide nitrogen species to the (++) quadrant in the (15)N spectrum. It is suggested that these quantitative data may be used in future functional and theoretical studies on the mammalian mitoNEET [2Fe-2S] cluster system.

Crystallization and preliminary X-ray diffraction studies of the prototypal homologue of mitoNEET (Tth-NEET0026) from the extreme thermophile Thermus thermophilus HB8.

MitoNEET (a mammalian mitochondrial outer membrane protein) is a potential pharmacological and clinical target of the insulin-sensitizer pioglitazone. The thermophilic homologue of mitoNEET (TTHA0026) from Thermus thermophilus HB8 has been heterologously overproduced in Escherichia coli and purified as a water-soluble prototypal protein containing the mitoNEET-like [2Fe-2S] cluster. The resultant recombinant protein, named Tth-NEET0026, has been crystallized in its oxidized form by the hanging-drop vapour-diffusion method using 17%(w/v) polyethylene glycol 4000, 8.5%(v/v) 2-propanol, 15%(v/v) glycerol and 0.085 M HEPES-NaOH pH 7.2. The dark reddish crystals diffracted to 1.80 A resolution and belonged to the tetragonal space group P4(3)2(1)2, with unit-cell parameters a = 45.51, c = 84.26 A. The asymmetric unit contains one protein molecule.

Crystallization and preliminary X-ray diffraction studies of a hyperthermophilic Rieske protein variant (SDX-triple) with an engineered rubredoxin-like mononuclear iron site.

In place of the Rieske [2Fe-2S] cluster, an archetypal mononuclear iron site has rationally been designed into a hyperthermophilic archaeal Rieske [2Fe-2S] protein (sulredoxin) from Sulfolobus tokodaii by three residue replacements with reference to the Pyrococcus furiosus rubredoxin sequence. The resulting sulredoxin variant, SDX-triple (H44I/A45C/H64C), has been purified and crystallized by the hanging-drop vapour-diffusion method using 65%(v/v) 2-methyl-2,4-pentanediol, 0.025 M citric acid and 0.075 M sodium acetate trihydrate pH 4.3. The crystals diffract to 1.63 A resolution and belong to the triclinic space group P1, with unit-cell parameters a = 43.56, b = 76.54, c = 80.28 A, alpha = 88.12, beta = 78.82, gamma = 73.46 degrees. The asymmetric unit contains eight protein molecules.

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 bc(1)/b(6)f complex is in part associated with the redox potential (E(m)) 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 (S(t)) and Tyr-Oeta (as with the Y156W variant, which has a modified histidine N(epsilon) pK(a,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-S(b/t) stretching vibrations. This is not observed in the case of the hydrogen bond between the bridging sulfide ligand (S(b)) and Ser-Ogamma, which is weak and/or unfavorably oriented for extensive coupling with the Fe-S(b/t) stretching vibrations.

15N HYSCORE characterization of the fully deprotonated, reduced form of the archaeal Rieske [2Fe-2S] center.

The hyperfine couplings for strongly and weakly coupled 15N nuclei around a reduced Rieske [2Fe-2S] center of uniformly 15N-labeled, hyperthermostable archaeal Rieske protein at pH 13.3 were determined by hyperfine sublevel correlation (HYSCORE) spectroscopy and compared with those at physiological pH. Significant changes in the hyperfine couplings of the terminal histidine Ndelta ligands and Nepsilon nuclei were observed between them, which can be explained by not only the redistribution of the unpaired electron spin density over the ligands but also the difference in the mixed-valence state of the fully deprotonated, reduced cluster. These quantitative data can be used in theoretical analysis for the selection of an appropriate model of the mixed-valence state of the reduced Rieske center at very alkaline pH.

Rational design of a mononuclear metal site into the archaeal Rieske-type protein scaffold.

Proteins containing Rieske-type [2Fe-2S] clusters play essential functions in all three domains of life. We engineered the two histidine ligands to the Rieske-type [2Fe-2S] cluster in the hyperthermophilic archaeal Rieske-type ferredoxin from Sulfolobus solfataricus to modify types and spacing of ligands and successfully converted the metal and cluster type at the redox-active site with a minimal structural change to a native Rieske-type protein scaffold. Spectroscopic analyses unambiguously established a rubredoxin-type mononuclear Fe3+/2+ center at the engineered local metal-binding site (Zn2+ occupies the iron site depending on the expression conditions). These results show the importance of types and spacing of ligands in the in vivo cluster recognition/insertion/assembly in biological metallosulfur protein scaffolds. We suggest that early ligand substitution and displacement events at the local metal-binding site(s) might have primarily allowed the metal and cluster type conversion in ancestral redox protein modules, which greatly enhanced their capabilities of conducting a wide range of unique redox chemistry in biological electron transfer conduits, using a limited number of basic protein scaffolds.

Orientation-selected 15N-HYSCORE detection of weakly coupled nitrogens around the archaeal rieske [2Fe-2S] center.

The weakly coupled 15N atoms around a reduced Rieske [2Fe-2S] cluster of the uniformly 15N-labeled, hyperthermostable archaeal Rieske protein appear to produce readily observable cross-peaks in the HYSCORE spectra, with the well-resolved couplings of 0.3-0.4 MHz for the Nepsilon and 1.1 MHz for the peptide backbone nitrogens, in addition to the contributions from the coordinated Ndelta atoms. These features can be used for structure-mechanism studies of the biological redox protein system involving the weakly coupled nitrogens in coupled electron-proton transfer reactions.

Crystallization and preliminary X-ray diffraction studies of the hyperthermophilic archaeal sulredoxin having the unique Rieske [2Fe-2S] cluster environment.

The hyperthermophilic archaeal sulredoxin from Sulfolobus tokodaii is a water-soluble high-potential Rieske [2Fe-2S] protein with unique pH-dependent redox properties compared with its mesophilic homologues in cytochrome bc1/b6f complexes. The oxidized recombinant sulredoxin has been crystallized by the hanging-drop vapour-diffusion method using 30%(v/v) polyethylene glycol 400, 0.1 M cadmium chloride and 0.1 M sodium acetate pH 4.6. The crystals diffracted to beyond 2.0 A resolution and belong to the cubic space group F4(1)32, with unit-cell parameter a = 163.00 +/- 0.05 A. The asymmetric unit contains one sulredoxin molecule. Three-wavelength MAD data were collected.

A comparative, two-dimensional 14N ESEEM characterization of reduced [2Fe-2S] clusters in hyperthermophilic archaeal high- and low-potential Rieske-type proteins.

Proteins of the Rieske and Rieske-type family contain a [2Fe-2S] cluster with mixed ligation by two histidines and two cysteines, and play important roles in various biological electron transfer reactions. We report here the comparative orientation-selected ESEEM and HYSCORE studies of the reduced clusters from two hyperthermophilic Rieske-type proteins; a high-potential, archaeal Rieske protein called sulredoxin (SDX) from Sulfolobus tokodaii with weak homology to the cytochrome bc-associated Rieske proteins, and a low-potential, archaeal homolog of an oxygenase-associated Rieske-type ferredoxin (ARF) from Sulfolobus solfataricus. (14)N ESEEM and HYSCORE spectra of SDX and ARF show well-defined variations, which are primarily determined by changes of quadrupole couplings (up to 50% depending on the selected orientation) of the two coordinated nitrogens. These are due to variations in coordination geometry of the histidine imidazole ligands rather than to variations of hyperfine couplings of these nitrogens, which do not exceed 8-10%. The measured quadrupole couplings and their differences in the two proteins are consistent with those calculated using the reported crystal structures of high- and low-potential Rieske proteins. These results suggest that exploration of quadrupole tensors might provide a more accurate method for characterization of the histidine coordination in different proteins and mutants than hyperfine tensors, and might have potential applications in a wider range of biological systems.

Characterization of the pH-dependent resonance Raman transitions of archaeal and bacterial Rieske [2Fe-2S] proteins.

The pH-dependent resonance Raman (RR) spectral changes of the cytochrome bc1-associated, high-potential Rieske proteins have frequently been invoked to explain the redox-linked ionization behavior. We report herein RR spectral data of archaeal and bacterial Rieske proteins that directly demonstrate the pH-dependent changes near and above pKa,ox2, but not around pKa,ox1, of the visible circular dichroism (CD) transitions. The RR spectral changes are attributed to modification of the immediate [2Fe-2S] cluster environment due to deprotonation of some exchangeable amide groups in the polypeptide backbone, rather than previously assumed simple changes of the Fe-Nimid stretching vibrations.

Engineering a three-cysteine, one-histidine ligand environment into a new hyperthermophilic archaeal Rieske-type [2Fe-2S] ferredoxin from Sulfolobus solfataricus.

We heterologously overproduced a hyperthermostable archaeal low potential (E(m) = -62 mV) Rieske-type ferredoxin (ARF) from Sulfolobus solfataricus strain P-1 and its variants in Escherichia coli to examine the influence of ligand substitutions on the properties of the [2Fe-2S] cluster. While two cysteine ligand residues (Cys(42) and Cys(61)) are essential for the cluster assembly and/or stability, the contributions of the two histidine ligands to the cluster assembly in the archaeal Rieske-type ferredoxin appear to be inequivalent as indicated by much higher stability of the His(64) --> Cys variant (H64C) than the His(44) --> Cys variant (H44C). The x-ray absorption and resonance Raman spectra of the H64C variant firmly established the formation of a novel, oxidized [2Fe-2S] cluster with one histidine and three cysteine ligands in the archaeal Rieske-type protein moiety. Comparative resonance Raman features of the wild-type, natural abundance and uniformly (15)N-labeled ARF and its H64C variant showed significant mixing of the Fe-S and Fe-N stretching characters for an oxidized biological [2Fe-2S] cluster with partial histidine ligation.

X-ray absorption spectroscopic analysis of reductive [2Fe-2S] cluster degradation in hyperthermophilic archaeal succinate:caldariellaquinone oxidoreductase subunits.

The biological [2Fe-2S] clusters play important roles in electron transfer and cellular signaling for a variety of organisms from archaea, bacteria to eukarya. The two recombinant hyperthermophilic archaeal [2Fe-2S] cluster-binding proteins, SdhC and the N-terminal domain fragment of SdhB, of Sulfolobus tokodaii respiratory complex II overproduced in Escherichia coli are thermostable as isolated, but moderately sensitive to reduction with excess dithionite. We used iron K-edge X-ray absorption spectroscopy to monitor the structural changes of their Fe sites in the irreversible [2Fe-2S] cluster degradation process. Regardless of the differences in the cluster-ligating cysteine motifs and the XAS-detectable [2Fe-2S](2+) cluster environments, a complete reductive breakdown of the [2Fe-2S] clusters resulted in the appearance of a new Fourier transform (FT) peak at approximately 3.3 A with a concomitant loss of the Fe-Fe interaction at ca. 2.7 A for both proteins. On the basis of the unambiguous assignment of the 3.3 A FT peak, our results suggest that a biological [2Fe-2S] cluster breakdown under reducing conditions generally releases Fe(2+) from the polypeptide chain into the aqueous solution, and the Fe(2+) might then be recruited as a secondary ferrous iron source for de novo biosynthesis and/or regulation of iron-binding enzymes in the cellular system.

Redox-dependent structural changes in archaeal and bacterial Rieske-type [2Fe-2S] clusters.

Proteins containing Rieske-type [2Fe-2S] clusters play important roles in many biological electron transfer reactions. Typically, [2Fe-2S] clusters are not directly involved in the catalytic transformation of substrate, but rather supply electrons to the active site. We report herein X-ray absorption spectroscopic (XAS) data that directly demonstrate an average increase in the iron-histidine bond length of at least 0.1 A upon reduction of two distantly related Rieske-type clusters in archaeal Rieske ferredoxin from Sulfolobus solfataricus strain P-1 and bacterial anthranilate dioxygenases from Acinetobacter sp. strain ADP1. This localized redox-dependent structural change may fine tune the protein-protein interaction (in the case of ARF) or the interdomain interaction (in AntDO) to facilitate rapid electron transfer between a lower potential Rieske-type cluster and its redox partners, thereby regulating overall oxygenase reactions in the cells.

Novel [2Fe-2S]-type redox center C in SdhC of archaeal respiratory complex II from Sulfolobus tokodaii strain 7.

The SdhC subunit of the archaeal respiratory complex II (succinate:quinone oxidoreductase) from Sulfolobus tokodaii strain 7 has a novel cysteine rich motif and is also related to archaeal and bacterial heterodisulfide reductase subunits. We overexpressed the sdhC gene heterologously in Escherichia coli and characterized the gene product in greater detail. Low temperature resonance Raman and x-ray absorption spectroscopic investigation collectively demonstrate the presence of a [2Fe-2S] cluster core with complete cysteinyl ligation (Center C) and an isolated zinc site in the recombinant SdhC. The [2Fe-2S]2+ cluster core is sensitive to dithionite, resulting in irreversible breakdown of the Fe-Fe interaction. EPR analysis confirmed that the novel Center C is an inherent redox center in the archaeal complex II, showing unique EPR signals in the succinate-reduced state. Distinct subunit and cofactor arrangements in the S. tokodaii respiratory complex II, as compared with those in mitochondrial and some mesophilic bacterial enzymes, indicate modular evolution of this ubiquitous electron entry site in the respiratory chains of aerobic organisms.