Stopped-flow mixing of the Desulfovibrio vulgaris two-iron superoxide reductase (2Fe-SOR) containing the ferrous active site with superoxide generates a dead time intermediate whose absorption spectrum is identical to that of a putative ferric-hydroperoxo intermediate previously observed by pulse radiolysis. The dead time intermediate is shown to be a product of reaction with superoxide and to be generated at a much higher proportion of active sites than by pulse radiolysis. This intermediate decays smoothly to the resting ferric active site ( approximately 30 s-1 at 2 degrees C and pH 7) with no other detectable intermediates. Deuterium isotope effects demonstrate that solvent proton donation occurs in the rate-determining step of dead time intermediate decay and that neither of the conserved pocket residues, Glu47 or Lys48, functions as a rate-determining proton donor between pH 6 and pH 8. Fluoride, formate, azide, and phosphate accelerate decay of the dead time intermediate and for azide or fluoride lead directly to ferric-azido or -fluoro complexes of the active site, which inhibit Glu47 ligation. A solvent deuterium isotope effect is observed for the azide-accelerated decay, and the decay rate constants are proportional to the concentrations and pKa values of HX (X- = F-, HCO2-, N3-). These data indicate that the protonated forms of the anions function analogously to solvent as general acids in the rate-determining step. The results support the notion that the ferrous SOR site reacts with superoxide by an inner sphere process, leading directly to the ferric-hydroperoxo intermediate, and demonstrate that the decay of this intermediate is subject to both specific- and general-acid catalysis.

We herein report the synthesis of 2‐deoxy‐2‐iodo‐glycosylamides, glycosylurea, N‐glycosylbenzotriazole, and N‐glycosyl imidazole by addition reaction of trimethylsilyl amides, imidazoles, and benzotriazoles to D‐glucal in the presence of N‐iodosuccinimide and propionitrile at 0 °C. Two diastereomers were isolated ― the α‐mannose and β‐gluco isomers. Reduction and substitution reaction of the iodine at position C‐2 led to formation of various 2‐deoxy‐N‐glycosides and 2‐hydroxy‐β‐D‐glucopyranosylamide. The newly generated compounds were screened for their inhibitory activity against various enzymes that included Nav1.7 sodium ion voltage‐gated channel in HEK293 cells and the results are discussed.

Addition of iron salts to chaotrope-denatured aporubredoxin (apoRd) leads to nearly quantitative recovery of its single Fe(SCys) 4 site and native protein structure without significant dilution of the chaotrope. This "high chaotrope" approach was used to examine iron binding and protein folding events using stopped-flow UV/vis absorption and CD spectroscopies. At 100-fold molar excess ferrous iron over denatured apoRd maintained in 5 M urea, the folded holoFe III Rd structure was recovered in >90% yield with t 1/2 < 10 msec. More modest excesses of iron also gave nearly quantitative holoRd formation in 5 M urea but with chronological resolution of iron binding and protein folding events. The results indicate structural recovery in 5 M urea consists of the minimal sequence: (1) binding of ferrous iron to the unfolded apoRd, (2) rapid formation of a near-native ferrous Fe(SCys) 4 site within a protein having no detectable secondary structure, (3) recovery of the ferrous Fe(SCys) 4 site chiral environment nearly concomitantly with (4) recovery of the native protein secondary structure. The rate of step 2 (and by inference, step 1) was not saturated even at 100-fold molar excess of iron. Analogous results obtained on Cys→Ser iron ligand variants support formation of an unfolded-Fe(SCys) 3 complex between steps 1 and 2, which we propose is the key nucleation event that pulls distal regions of the protein chain together. These results show that folding of chaotrope-denatured apoRd is iron-nucleated and driven by extraordinarily rapid formation of the Fe(SCys) 4 site from an essentially random coil apoprotein. This high chaotrope, multi-spectroscopic approach could clarify folding pathways of other [M(SCys) 3 Supporting Information AvailableNear UV-CD spectra of apo and holoRds in high urea, plots of midpoint urea denaturations of apoRds, spectral time courses of ferrous ammonium sulfate oxidations in 5 M urea, static far-UV CD spectra of apo and holoRds in high urea, semi-log plots of the time courses in Figure 3 and Figure 6, stopped-flow absorption spectral time course for apoRd +110-fold molar excess iron in 4.6 M urea. This material is available free of charge via the Internet at http://pubs.acs.org. 1 Abbreviations used: Rd, rubredoxin, apoRd, metal-free Rd; holoRd, iron-containing Rd in its native folded structure; Cp, Clostridium pasteurianum; CD, circular dichroism; wt, wild type; Tris-HCl, tris(hydroxymethyl)aminomethane-hydrochloride; CXXC, two cysteine residues (C) separated by two other residues, X, in the Rd amino acid sequence; C6S, C9S, C39S, C42S, Cys→Ser variant Rds; unfolded-Fe II (SCys) 4 , species exhibiting near-UV absorption characteristic of the native Fe II (SCys) 4 site but no CD signal for protein secondary structure; chiral-Fe II (SCys) 4 , species exhibiting near-UV absorption and CD signals characteristic of the native Fe II (SCys) 4 site; folded-Fe II (SCys) 4 , species exhibiting absorption and CD signals for the Fe II (SCys) 4 site and protein secondary structure CD signal char...

The ribozyme from bacterial ribonuclease P recognizes two structural modules in a tRNA substrate: the T stem-loop and the acceptor stem. These two modules are connected through a helical linker. The T stem-loop binds at a surface confined in a folding domain away from the active site. Substrates for the Bacillus subtilis RNase P RNA were previously selected in vitro that are shown to bind comparably well or better than a tRNA substrate. Chemical modification of P RNA-substrate complexes with dimethylsulfate and kethoxal was performed to determine how the P RNA recognizes three in vitro selected substrates. All three substrates bind at the surface known to interact with the T stem-loop of tRNA. Similar to a tRNA, the secondary structure of these substrates contains a helix around the cleavage site and a hairpin loop at the corresponding position of the T stem-loop. Unlike a tRNA, these two structural modules are connected through a non-helical linker. The two structural modules in the tRNA and in the selected substrates bind to two different domains in P RNA. The properties of substrate recognition exhibited by this ribozyme may be exploited to isolate new ribozyme-substrate pairs with interactive structural modules.