PubMed · 5811900
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B Sheard. 1969-09-06. Internal mobility in phospholipids.. https://doi.org/10.1038/2231057a0
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The active site of naphthalene 1,2-dioxygenase (NDO) contains a Rieske Fe-S cluster and a mononuclear non-heme iron, which are contributed by different alpha-subunits in the (alphabeta)(3) structure. The enzyme catalyzes cis-dihydroxylation of aromatic substrates, in addition to numerous other adventitious oxidation reactions. High-resolution Mims (2)H-ENDOR (electron nuclear double resonance) spectra have been recorded for the NO-ferrous center of NDO bound with the substrates d(8)-naphthalene, d(2)-naphthalene, d(8)-toluene, d(3)-toluene, and d(6)-benzene; samples were prepared in a D(2)O buffer to test for solvent-derived ligands; spectra were collected for enzymes with the Rieske diiron center in both its oxidized and reduced states. A sharp quartet ENDOR pattern from a nearby deuteron of the substrate in a major binding geometry (denoted as A) was detected for all perdeuterated substrates. Examination of the sample prepared with 1,4-di-deutero-naphthalene shows that the signal arises from D1. Analysis of two-dimensional (2-D) orientation-selective ENDOR patterns collected for this sample defined the location of the D1 deuteron, with respect to the g-frame of the iron center and the orientation of the C-D1 bond. Consideration of the orientations of naphthalene that are permitted within the constraints of these results, as supported by a novel approach to simulations of orientation-selective, 2-D ENDOR patterns for the perdeuterated naphthalene sample, which summed contributions from D1/D2/D8, disclose the geometry of the naphthalene and the Fe-NO fragment. The two deuterons of the reactive carbons, D1 and D2, are closest to the Fe atom (r(Fe)(-)(D1) approximately 4.3 A, r(Fe)(-)(D2) approximately 5.0 A), whereas D8 is farther away (r(Fe)(-)(D8) approximately 5.3 A). Perhaps more instructive, D1-N and D2-N distances to the O(2) surrogate, NO, are approximately 2.4 and approximately 3.3 A, respectively, whereas the D8-N distance is approximately 3.7 A. The data show that benzene and the aromatic ring of toluene also sit within the substrate-binding pocket adjacent to the mononuclear Fe atom. These rings occupy a position similar to that of the "proximal" ring of naphthalene, with the closest ring deuteron being located at a distance of approximately 4.3-4.4 A from the Fe atom and with the Fe-D vector being slightly off the Fe-N(O) direction. In particular, comparison of the data for d(8)-toluene and methyl-d(3)-toluene shows that the methyl group of toluene points away from the Fe atom, despite observations that the oxidation of toluene occurs at the methyl group during catalysis. The Rieske cluster is reduced during both steady-state and single-turnover catalysis; therefore, the effect of its oxidation state on the geometry of substrate binding was examined. The spectra from the NDO-naphthalene complex also revealed a second binding conformation (denoted as B), in which the substrate is located approximately 0.5 A farther from the Fe atom. The relative populations of A- and B-sites are allosterically changed when the Rieske cluster is reduced. ENDOR of exchangeable protons shows that the water/hydroxide of Fe-NDO is retained upon binding NO.
[(2)H(10)]-4-Mercapto-4-methylpentan-2-one (d(10)-1), [(2)H(2)]-3-mercaptohexan-1-ol (d(2)-2), and [(2)H(5)]-3-mercaptohex-1-yl acetate (d(5)-3), deuterated analogues of impact odorants of wines, were used to determine quantitatively the natural compounds in white wines (Muscadet, Sauvignon, and Bacchus) with a stable isotope dilution assay using gas chromatography coupled either with ion trap tandem mass spectrometry (GC-ITMS-MS) or with atomic emission detection monitored on sulfur-selective acquisition (GC-AED). The thiol compounds were recovered from wines by liquid-liquid extraction, then purified from the wine extracts by covalent chromatography, and analyzed. The quantitative determination of 4-mercapto-4-methylpentan-2-one 1 in the wines that were analyzed was performed better with GC-AED than with GC-ITMS-MS under the conditions that were used. However, the detection limit of the method was higher than the odor threshold of 4-mercapto-4-methylpentan-2-one 1 in wine (5 vs 0.8 ng/L). The levels of this compound in the Sauvignon and Bacchus wines were much higher than its odor threshold, but it was not detectable in the Muscadet wines. On the contrary, GC-ITMS-MS was much more sensitive than GC-AED for detection of 3-mercaptohexan-1-ol 2 and 3-mercaptohex-1-yl acetate 3, and the detection limits were much lower than their odor thresholds in wine. The former compound was detected in all of the Muscadet wines that were analyzed at levels always higher than its odor detection threshold, while the latter occurred at levels higher than its odor threshold in only one Muscadet wine.
The electron-stimulated production of D2 from amorphous solid D2O deposited on Pt(111) is investigated as a function of film thickness. The D2 yield has two components that have distinct reaction kinetics. Using isotopically layered films of H2O and D2O demonstrates that the D2 is produced in reactions that occur at both the Pt/amorphous solid water (ASW) interface and the ASW/vacuum interface, but not in the bulk. The energy for the reactions, however, is absorbed in the bulk of the films and electronic excitations diffuse to the interfaces where they drive the reactions.