Characterization of the particulate methane monooxygenase metal centers in multiple redox states by X-ray absorption spectroscopy

The integral membrane enzyme particulate methane monooxygenase (pMMO) converts methane, the most inert hydrocarbon, to methanol under ambient conditions. The 2.8-A resolution pMMO crystal structure revealed three metal sites: a mononuclear copper center, a dinuclear copper center, and a nonphysiological mononuclear zinc center. Although not found in the crystal structure, solution samples of pMMO also contain iron. We have used X-ray absorption spectroscopy to analyze the oxidation states and coordination environments of the pMMO metal centers in as-isolated (pMMO(iso)), chemically reduced (pMMO(red)), and chemically oxidized (pMMO(ox)) samples. X-ray absorption near-edge spectra (XANES) indicate that pMMO(iso) contains both Cu(I) and Cu(II) and that the pMMO Cu centers can undergo redox chemistry. Extended X-ray absorption fine structure (EXAFS) analysis reveals a Cu-Cu interaction in all redox forms of the enzyme. The Cu-Cu distance increases from 2.51 to 2.65 A upon reduction, concomitant with an increase in the average Cu-O/N bond lengths. Appropriate Cu2 model complexes were used to refine and validate the EXAFS fitting protocols for pMMO(iso). Analysis of Fe EXAFS data combined with electron paramagnetic resonance (EPR) spectra indicates that Fe, present as Fe(III), is consistent with heme impurities. These findings are complementary to the crystallographic data and provide new insight into the oxidation states and possible electronic structures of the pMMO Cu ions.     

[M + 11]+˙ and [M + 11]−˙ ions in the nitrogen positive and negative ion chemical ionization mass spectra of aromatic amines

The N₂ negative ion chemical ionization (NICI) mass spectra of aniline, aminonaphthalenes, aminobiphenyls and aminoanthracenes show an unexpected addition appearing at [M + 11]^?˙. This addition is also observed in the N₂ positive chemical ionization (PCI) mass spectra. An ion at [M – 15]^? is found in the NICI spectra of aminoaromatics such as aniline, 1- and 2-aminonaphthalene and 1- and 2-aminoanthracene. Ion formation was studied using labeled reagents, variation of ion source pressure and temperature and examination of ion chromatograms. These experiments indicate that the [M + 11]^?˙, [M – 15]^?˙ and [M + 11]^+˙ ions result from the ionization of analytes altered by surface-assisted reactions. Experiments with ¹⁵N₂, [¹⁵N] aniline, [2,3,4,5,6-²H₅] aniline and [¹³C₆] aniline show that the [M + 11]^?˙ ion corresponds to [M + N – 3H]^?˙. The added nitrogen originates from the N₂ buffer gas and the addition occurs with loss of one ring and two amino group hydrogens. Fragmentation patterns in the N₂ PCI mass spectrum of aniline suggest that the neutral product of the surface-assisted reaction is 1,4-dicyanobuta-1,3-diene. Experiments with diamino-substituted aromatics show analogous reactions resulting in the formation of [M – 4H]^?˙ ions for aromatics with ortho-amino groups. Experiments with methylsubstituted aminoaromatics indicate that unsubstituted sites ortho to the amino group facilitate nitrogen addition, and that methyl groups provide additional sites for nitrogen addition.     

Asymmetric enamide hydrogenation using planar-chiral cyrhetrenes

The catalytic asymmetric hydrogenation of α-arylenamides using catalysts prepared in situ from [Rh(cod)₂]BF₄ and cyrhetrenyldiphosphines was effective with a range of enamides. The corresponding acetamides were obtained with up to 93% ee.     

Negative ions generated by reactions with oxygen in the chemical ionization source. I. Characterization of gas-phase and wall-catalyzed reactions of fluorene,anthracene and fluoranthene

The methane negative ion chemical ionization (NICI) mass spectra of polycyclic aromatic hydrocarbons are usually dominated by molecular, M^{? ˙} or M ? H^? ions; however, ions resulting from additions to M have also been reported. Some of these ions have been observed at [M + 14]^{? ˙}, [M + 15]^?, [M + 30]^? and [M + 32]^?˙ and have been attributed to reactions with either oxygen-containing impurities in the buffer gas or alkyl radical species generated by ionization of a hydrocarbon buffer gas. In this study, the NICI spectra of fluorene, anthracene and fluoranthene were studied in detail using quadrupole and Fourier transform mass spectrometers. Spectra were acquired when reactive species such as oxygen, water, nitrous oxide and carbon dioxide were added to the nitrogen buffer gas. Experiments with deuterated methane were also carried out. These studies indicated that buffer gas impurities affect the NICI spectra; however, gas-phase ion-molecule reactions were not responsible for all of the observed products. In addition to electron- and ion-molecule reactions, ions were observed that resulted from wall-catalyzed oxidation reactions followed by electron capture. These reactions were enhanced by the addition of oxygen and elevated ion source temperatures. Depending upon the parent PAH structure, oxidation products such as ketones, quinones and anhydrides were formed.     

Total Synthesis of (R,R,R)‐α‐Tocopherol Through Asymmetric Cu‐Catalyzed 1,4‐Addition

By introducing a disposable activating substituent at C‐3, the asymmetric 1,4‐addition to a notoriously unreactive 2‐substituted chromenone was achieved with high levels of (2R)‐stereoselectivity in the presence of a chiral Cu^I‐phosphoramidite complex as a catalyst. This paved the way for an efficient and conceptually novel synthesis of (R,R,R)‐α‐tocopherol from readily available starting materials.     

Circular dichroism (CD) spectroscopy of the assembly and disassembly of SNAREs: The proteins involved in membrane fusion in cells

In this study, we report for the first time that both t-SNAREs and v-SNARE and their complexes in buffered suspension, exhibit defined peaks at CD signals of 208 and 222 nm wavelengths, consistent with a higher degree of helical secondary structure. Surprisingly, when incorporated in lipid membrane, both SNAREs and their complexes exhibit reduced folding. In presence of NSF–ATP, the SNARE complex disassembles, as reflected from the CD signals demonstrating elimination of α-helices within the structure.     

Investigation of the copper binding site and the role of histidine as a ligand in riboflavin binding protein

Riboflavin Binding Protein (RBP) binds copper in a 1:1 molar ratio, forming a distinct well-ordered type II site. The nature of this site has been examined using X-ray absorption and pulsed electron paramagnetic resonance (EPR) spectroscopies, revealing a four coordinate oxygen/nitrogen rich environment. On the basis of analysis of the Cambridge Structural Database, the average protein bound copper-ligand bond length of 1.96 A, obtained by extended x-ray absorption fine structure (EXAFS), is consistent with four coordinate Cu(I) and Cu(II) models that utilize mixed oxygen and nitrogen ligand distributions. These data suggest a Cu-O 3N coordination state for copper bound to RBP. While pulsed EPR studies including hyperfine sublevel correlation spectroscopy and electron nuclear double resonance show clear spectroscopic evidence for a histidine bound to the copper, inclusion of a histidine in the EXAFS simulation did not lead to any significant improvement in the fit.     

Human frataxin: iron and ferrochelatase binding surface

The coordinated iron structure and ferrochelatase binding surface of human frataxin have been characterized to provide insight into the protein's ability to serve as the iron chaperone during heme biosynthesis.