Crystal structure of RumA, an iron-sulfur cluster containing E. coli ribosomal RNA 5-methyluridine methyltransferase

RumA catalyzes transfer of a methyl group from S-adenosylmethionine (SAM) specifically to uridine 1939 of 23S ribosomal RNA in Escherichia coli to yield 5-methyluridine. We determined the crystal structure of RumA at 1.95 A resolution. The protein is organized into three structural domains: The N-terminal domain contains sequence homology to the conserved TRAM motif and displays a five-stranded beta barrel architecture characteristic of an oligosaccharide/oligonucleotide binding fold. The central domain contains a [Fe(4)S(4)] cluster coordinated by four conserved cysteine residues. The C-terminal domain displays the typical SAM-dependent methyltransferase fold. The catalytic nucleophile Cys389 lies in a motif different from that in DNA 5-methylcytosine methyltransferases. The electrostatic potential surface reveals a predominately positively charged area that covers the concave surface of the first two domains and suggests an RNA binding mode. The iron-sulfur cluster may be involved in the correct folding of the protein or may have a role in RNA binding.     

Organization of the ribosomal RNA gene cluster in Aspergillus nidulans

DNA coding for ribosomal RNA in Aspergillus nidulans was found to consist of a unit 7.8 kb in size which is tandemly repeated in the genome and codes for 5.8S, 18S and 26S rRNA. The repeat unit has been cloned, and its restriction map and the location of the individual rRNA coding sequences within the unit have been established.     

EPR studies of iron-sulfur clusters in isolated subunits and subfractions of NADH-ubiquinone oxidoreductase

The thermodynamic and EPR characteristics of the iron-sulfur clusters of NADH-ubiquinone oxidoreductase have been examined in various subfractions and subunits of the enzyme. These were obtained by fragmentation of the enzyme with chaotropic agents and detergent and salt fractionation. We provide evidence for the presence of three tetranuclear clusters and five or six binuclear clusters, accounting well for the chemically determined iron content of this enzyme (22-24 atoms/molecule of FMN). Some of the clusters can be identified with EPR-detectable species in intact NADH-ubiquinone oxidoreductase and, by combining information on subunit topography and spin-spin interactions between redox centers in the native enzyme, we propose a tentative scheme for the spatial organization of these iron-sulfur clusters in the enzyme and in the membrane.     

Carotenoid radical cations and dications: EPR, optical, and electrochemical studies

Investigations of the structure and properties of paramagnetic carotenoid radical cations and diamagnetic carotenoid dications using electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR) spectroscopy in conjunction with electrochemical, optical, and HPLC measurements, and molecular orbital calculations are described. These methods were applied to determine how the carotenoid radical cations and dications can be formed, their electron-transfer properties and stability in various media, and the mechanism by which carotenoid radical cations can isomerize.     

EPR studies on a Hipip type iron-sulfur center in the succinate dehydrogenase segment of the respiratory chain

In addition to the two species of ferredoxin-type iron-sulfur centers (Centers S-1 and S-2), a Hipip-type iron-sulfur center (Center S-3) has been detected in the reconstitutively active soluble succinate dehydrogenases. E_m7,4 determined in a particulate, antimycin A sensitive succinate-cytochrome $\text{c}$ reductase is +60 ± 15 mV. This center is extremely labile towards oxygen in a manner similar to the reconstitutive activity of the dehydrogenase. Even freshly prepared reconstitutively active enzyme shows a considerably diminished content of Center S-3 relative to flavin and displays a partly modified spectra. All reconstitutively inactive dehydrogenases give rise to a highly modified or no Center S-3 spectra at all. These observations indicate that Center S-3 is a constituent of succinate dehydrogenase and plays a role in the physiological function of the enzyme, $\text{i.e.}$ transferring electrons most probably to ubiquinone.     

Identification of the iron-sulfur center of spinach ferredoxin-nitrite reductase as a tetranuclear center, and preliminary EPR studies of mechanism.

EPR spectroscopic and chemical analyses of spinach nitrite reductase show that the enzyme contains one reducible iron-sulfur center, and one site for binding either cyanide or nitrite, per siroheme. The heme is nearly all in the high spin ferric state in the enzyme as isolated. The extinction coefficient of the enzyme has been revised to E386 = 7.6 X 10(4) cm-1 (M heme)-1. The iron-sulfur center is reduced with difficulty by agents such as reduced methyl viologen (equilibrated with 1 atm of H2 at pH 7.7 in the presence of hydrogenase) or dithionite. Complexation of the enzyme with CO (a known ligand for nitrite reductase heme) markedly increases the reducibility of the iron-sulfur center. New chemical analyses and reinterpretation of previous data show that the enzyme contains 6 mol of iron and 4 mol of acid-labile S2-/mol of siroheme. The EPR spectrum of reduced nitrite reductase in 80% dimethyl sulfoxide establishes clearly that the enzyme contains a tetranuclear iron-sulfur (Fe4S4) center. The ferriheme and Fe4S4 centers are reduced at similar rates (k = 3 to 4 s-1) by dithionite. The dithionite-reduced Fe4S4 center is rapidly (k = 100 s-1) reoxidized by nitrite. These results indicate a role for the Fe4S4 center in catalysis.     

EPR studies on two ferredoxin-type iron-sulfur centers in reconstitutively active, inactive, and reactivated soluble succinate dehydrogenases

Two distinct iron-sulfur centers, S-1 and S-2 are present in both reconstitutively active and inactive soluble succinate dehydrogenase preparations in approximately equivalent concentrations to that of bound flavin. The midpoint potentials at pH 7.4 of these centers are ?5 ± 15 mV and ?400 ± 15 mV, respectively. EPR characteristics of Center S-2, observed above 6° K, are not significantly different in the active and inactive dehydrogenases. At lower temperatures, however, major line shape modifications of Center S-2 spectra are observed in the reconstitutively inactive dehydrogenases, but neither in the active dehydrogenase nor in the particulate preparations. This phenomenon may reflect spin-spin interaction between Centers S-1 and S-2. Chemical reactivation of the reconstitutively inactive preparations abolishes this resonance modification and restores the normal line shape. This is a demonstration of another close correlation between a physical property and reconstitutive activity of succinate dehydrogenase.     

Organization of the ribosomal RNA gene cluster in the yeast Saccharomyces cerevisiae

The chromosomal organization of the ribosomal RNA gene cluster from Saccharomyces cerevisiae was investigated. 18 S rRNA R-loops were formed with unfractionated high molecular weight DNA crosslinked once per 2.7 × 10³ bases with trioxsalen and observed in the electron microscope. Almost all the R-loops were found in very long continuous 9.34 ± 0.18 × 10³ base repeating units. In addition, molecules were found at a frequency of one to two per genome equivalent of rDNA where several rRNA genes were linked to long stretches of non-rDNA. These results suggest that rDNA is arranged in a single tandem repetitive cluster of 100 to 140 genes flanked on one or both sides by non-rDNA.     

Recognition of nucleotide G745 in 23 S ribosomal RNA by the rrmA methyltransferase

Methylation of the N1 position of nucleotide G745 in hairpin 35 of Escherichia coli 23 S ribosomal RNA (rRNA) is mediated by the methyltransferase enzyme RrmA. Lack of G745 methylation results in reduced rates of protein synthesis and growth. Addition of recombinant plasmid-encoded rrmA to an rrmA-deficient strain remedies these defects. Recombinant RrmA was purified and shown to retain its activity and specificity for 23 S rRNA in vitro. The recombinant enzyme was used to define the structures in the rRNA that are necessary for the methyltransferase reaction. Progressive truncation of the rRNA substrate shows that structures in stem-loops 33, 34 and 35 are required for methylation by RrmA. Multiple contacts between nucleotides in these stem-loops and RrmA were confirmed in footprinting experiments. No other RrmA contact was evident elsewhere in the rRNA. The RrmA contact sites on the rRNA are inaccessible in ribosomal particles and, consistent with this, 50 S subunits or 70 S ribosomes are not substrates for RrmA methylation. RrmA resembles the homologous methyltransferase TlrB (specific for nucleotide G748) as well as the Erm methyltransferases (nucleotide A2058), in that all these enzymes methylate their target nucleotides only in the free RNA. After assembly of the 50 S subunit, nucleotides G745, G748 and A2058 come to lie in close proximity lining the peptide exit channel at the site where macrolide, lincosamide and streptogramin B antibiotics bind.     

Studies on the spin-spin interaction between flavin and iron-sulfur cluster in an iron-sulfur flavoprotein

When the di- or trimethylamine dehydrogenases (trimethylamine:(acceptor) oxidoreductase (demethylating), EC 1.5.99.7) of certain methylotrophic bacteria are reduced by two electrons with substrate unusual EPR signals arise at g = 2 and g = 4 (Steenkamp, D.J. and Beinert, H. (1982) Biochem. J. 207, 233-239; 241-252) indicative of spin-spin interaction between the FMN and iron-sulfur compounds of these enzymes. An attempt is made to understand, describe and simulate these spectra in terms of a triplet state with possible contributions from both dipolar and anisotropic exchange (J) interactions. No direct measurement of J is available, but various approaches to setting limits to J are outlined. According to these, J approximately 0.4 to 3 cm-1 or 15 to 50 cm-1. The spectra show, in the g = 2 region, a pair of rather sharp inner and a pair of broad outer lines; the latter broaden as well as move out from the center with increasing time (after substrate addition) and substrate concentration, while there is little change of g = 4. The best fits to such spectra were obtained by assuming distribution of D and E values, depending on substrate effects and arriving presumably from 'g-strain'. The fact that both shapes and intensities at g = 2 and g = 4 could be reproduced simultaneously at two frequencies indicates that the assumptions underlying our approaches and interpretations are permissible and reasonable, although we cannot claim their uniqueness. The distance between the centers of the spin densities of the flavin radical and the Fe-S cluster is thought to lie between the limits 3 to 5 A if the asymmetries in the spin-spin interaction are magnetic dipole-dipole in origin. Because there is an indication that the interaction is anisotropic exchange, the upper limit is less stringent.     

Biotin synthase contains two distinct iron-sulfur cluster binding sites: chemical and spectroelectrochemical analysis of iron-sulfur cluster interconversions.

Biotin synthase is an iron-sulfur protein that utilizes AdoMet to catalyze the presumed radical-mediated insertion of a sulfur atom between the saturated C6 and C9 carbons of dethiobiotin. Biotin synthase (BioB) is aerobically purified as a dimer that contains [2Fe-2S](2+) clusters and is inactive in the absence of additional iron and reductants, and anaerobic reduction of BioB with sodium dithionite results in conversion to enzyme containing [4Fe-4S](2+) and/or [4Fe-4S](+) clusters. To establish the predominant cluster forms present in biotin synthase in anaerobic assays, and by inference in Escherichia coli, we have accurately determined the extinction coefficient and cluster content of the enzyme under oxidized and reduced conditions and have examined the equilibrium reduction potentials at which cluster reductions and conversions occur as monitored by UV/visible and EPR spectroscopy. In contrast to previous reports, we find that aerobically purified BioB contains ca. 1.2-1.5 [2Fe-2S](2+) clusters per monomer with epsilon(452) = 8400 M(-)(1) cm(-)(1) per monomer. Upon reduction, the [2Fe-2S](2+) clusters are converted to [4Fe-4S] clusters with two widely separate reduction potentials of -140 and -430 mV. BioB reconstituted with excess iron and sulfide in 60% ethylene glycol was found to contain two [4Fe-4S](2+) clusters per monomer with epsilon(400) = 30 000 M(-)(1) cm(-)(1) per monomer and is reduced with lower midpoint potentials of -440 and -505 mV, respectively. Finally, as predicted by the measured redox potentials, enzyme incubated under typical anaerobic assay conditions is repurified containing one [2Fe-2S](2+) cluster and one [4Fe-4S](2+) cluster per monomer. These results indicate that the dominant stable cluster state for biotin synthase is a dimer containing two [2Fe-2S](2+) and two [4Fe-4S](2+) clusters.     

Inclusion complexes of carotenoids with cyclodextrins: 1H NMR, EPR, and optical studies

Direct evidence of carotenoid/cyclodextrin inclusion complex formation was obtained for the water-soluble sodium salt of beta-caroten-8'-oic acid (IV) by using 1H NMR and UV-Vis absorption spectroscopy. It was shown that this carotenoid forms a stable 1:1 inclusion complex with beta-cyclodextrin (stability constant K11 approximately 1500 M(-1)). All other carotenoids under study in the presence of cyclodextrins (CDs) form large aggregates in aqueous solution as demonstrated by very broad absorption spectra and considerable change in color. By using the EPR spin trapping technique, the scavenging ability of IV toward OOH radicals was compared in DMSO and in the aqueous CD solution. A considerable decrease in PBN/OOH spin adduct yield was detected in the presence of uncomplexed IV because of a competing reaction of the carotenoid with OOH radical. No such decrease occurred in the presence of the IV/CD complex. Moreover, a small increase in spin adduct yield (pro-oxidant effect) is most likely due to the reaction of the carotenoid with Fe3+ to regenerate Fe2+, which in turn regenerates the OOH radical. Our data show that CD protects the carotenoid from reactive oxygen species. On the other hand, complexation with CD results in considerable decrease in antioxidant ability of the carotenoid.     

Human ribosomal RNA gene cluster: identification of the proximal end containing a novel tandem repeat sequence

Human ribosomal RNA genes (rDNA) are arranged as tandem repeat clusters on the short arms of five pairs of acrocentric chromosomes. We have demonstrated that a majority of the rDNA clusters are detected as 3-Mb DNA fragments when released from human genomic DNA by EcoRV digestion. This indicated the absence of the EcoRV restriction site within the rDNA clusters. We then screened for rDNA-positive cosmid clones using a chromosome 22-specific cosmid library that was constructed from MboI partial digests of the flow-sorted chromosomes. Three hundred twenty rDNA-positive clones negative for the previously reported distal flanking sequence (pACR1) were chosen and subjected to EcoRV digestion. Seven clones susceptible to EcoRV were further characterized as candidate clones that might have been derived from the junctions of the 3-Mb rDNA cluster. We identified one clone containing part of the rDNA unit sequence and a novel flanking sequence. Detailed analysis of this unique clone revealed that the coding region of the last rRNA gene located at the proximal end of the cluster is interrupted with a novel sequence of 147 bp that is tandemly repeated and is connected with an intervening 68-bp unique sequence. This junction sequence was readily amplified from chromosomes 21 and 15 as well as 22 using the polymerase chain reaction. Fluorescence in situ hybridization further indicated that the 147-bp sequence repeat is commonly distributed among all the acrocentric short arms.