Binding sites of nitrogenase: kinetic and theoretical studies of cyanide binding to extracted FeMo-cofactor derivatives

The first kinetic study of a substrate (CN(-)) binding to the isolated active site (extracted FeMo-cofactor) of nitrogenase is described. The kinetics of the reactions between CN(-) and various derivatives of extracted FeMo-cofactor [FeMoco-L; where L is bound to Mo, and is NMF, Bu(t)NC, or imidazole (ImH)] have been followed using a stopped-flow, sequential-mix method in which the course of the reaction is followed indirectly, by monitoring the change in the rate of the reaction of the cofactor with PhS(-). The kinetic results, together with DFT calculations, indicate that the initial site of CN(-) binding to FeMoco-L is controlled by a combination of the electron-richness of the cluster core and lability of the Mo-L bond. Ultimately, the reactions between FeMoco-L and CN(-) involve displacement of L and binding of CN(-) to Mo. These reactions occur with a variety of rates and rate laws dependent on the nature of L. For FeMoco-NMF, the reaction with CN(-) is complete within the dead-time of the apparatus (ca. 4 ms), while with FeMoco-CNBu(t) the reaction is much slower and exhibits first order dependences on the concentrations of both FeMoco-CNBu(t) and CN(-) (k = 2.5 +/- 0.5 x 10(4) dm(3) mol(-1) s(-1)). The reaction of FeMoco-ImH with CN(-) occurs at a rate which exhibits a first order dependence on FeMoco-ImH but is independent of the concentration of CN(-) (k = 50 +/- 10 s(-1)). The results are interpreted in terms of CN(-) binding directly to the Mo site for FeMoco-NMF and FeMoco-ImH, but with FeMoco-CNBu(t) initial binding at an Fe site is followed by movement of CN(-) to Mo. Complementary DFT calculations are consistent with this interpretation, indicating that, in FeMoco-L, the Mo-L bond is stronger for L = ImH than for L = CNBu(t) and the binding of CN(-) to Mo is stronger than to any Fe atom in the cofactor.     

PHOTOINHIBITION OF PHOTOSYNTHESIS IN NATURAL WATERS*

Abstract— A quantitative analysis of the wavelength-dependent influence of solar irradiance on natural phytoplankton photosynthesis has been made. The effect on productivity due to several different UV radiation regimes has been measured. In the course of this analysis, it has been shown that the biological weighting function for photoinhibition of chloroplasts (Jones and Kok, 1966) allows the calculation of a biologically effective dose which is consistent with the measured photoinhibition in natural phytoplankton populations. The ecological implications of a change in available UV radiation, possibly due to anthropogenic altering of the ozone layer, are explored and it is found that the present static bottle ^l4C technique of measuring in situ phytoplankton productivity does not lend itself to assessing accurately the potential ecological consequences of possible increased MUV (middle ultraviolet radiation in the 280–340 nm region) on phytoplankton populations. A small change in MUV has a relatively minor effect on photoinhibition dose rates whereas it has a large potential effect on DNA dose rates.     

Disulfides of manganese carbonyl. Synthesis of Mn(2)(CO)(7)(mu-S2) and its reactions with tertiary phosphines and arsines

The reaction of Mn(2)(CO)(9)(NCMe) with thiirane yielded the sulfidomanganese carbonyl compounds Mn(2)(CO)(7)(mu-S(2)), 2, Mn(4)(CO)(15)(mu(3)-S(2))(mu(4)-S(2)), 3, and Mn(4)(CO)(14)(NCMe)(mu(3)-S(2))(mu(4)-S(2)), 4, by transfer of sulfur from the thiirane to the manganese complex. Compound 3 was obtained in better yield from the reaction of 2 with CO, and compound 4 is obtained from the reaction of 2 with NCMe. The reaction of 2 with PMe(2)Ph yielded the tetramanganese disulfide Mn(4)(CO)(15)(PMe(2)Ph)(2)(mu(3)-S)(2), 5, and S=PMe(2)Ph. The reaction of 5 with PMe(2)Ph yielded Mn(4)(CO)(14)(PMe(2)Ph)(3)(mu(3)-S)(2), 6, by ligand substitution. The reaction of 2 with AsMe(2)Ph yielded the new complexes Mn(4)(CO)(14)(AsMe(2)Ph)(2)(mu(3)-S(2))(2), 7, Mn(4)(CO)(14)(AsMe(2)Ph)(mu(3)-S(2))(mu(4)-S(2)), 8, Mn(6)(CO)(20)(AsMe(2)Ph)(2)(mu(4)-S(2))(3), 9, and Mn(2)(CO)(6)(AsMe(2)Ph)(mu-S(2)), 10. Reaction of 2 with AsPh(3) yielded the monosubstitution derivative Mn(2)(CO)(6)(AsPh(3))(mu-S(2)), 11. Reaction of 7 with PMe(2)Ph yielded Mn(4)(CO)(15)(AsMe(2)Ph)(2)(mu(3)-S)(2), 12. The phosphine analogue of 7, Mn(4)(CO)(14)(PMe(2)Ph)(2)(mu(3)-S(2))(2), 13, was prepared from the reaction of Mn(2)(CO)(9)(PMe(2)Ph) with Me(3)NO and thiirane. Compounds 2-9 and 11-13 were characterized by single-crystal X-ray diffraction. Compound 2 contains a disulfido ligand that bridges two Mn(CO)(3) groups that are joined by a Mn-Mn single bond, 2.6745(5) A in length. A carbonyl ligand bridges the Mn-Mn bond. Compounds 3 and 4 contain four manganese atoms with one triply bridging and one quadruply bridging disulfido ligand. Compounds 5 and 6 contain four manganese atoms with two triply bridging sulfido ligands. Compound 9 contains three quadruply bridging disulfido ligands imbedded in a cluster of six manganese atoms.     

Characterization of ginseng saponins using electrospray mass spectrometry and collision-induced dissociation experiments of metal-attachment ions

Electrospray mass spectrometry (ESMS) and collision-induced dissociation (CID) methodologies have been developed for the structural characterization of ginseng saponins (ginsenosides). Ginsenosides are terpene glycosides containing a triterpene core to which one to four sugars may be attached. They are neutral molecules which readily form molecular metal-attachment ions in positive ion ESMS experiments. In the presence of ammonium hydroxide intense deprotonated ions are generated. Both positive and negative ion ESMS experiments were found to be useful for molecular mass and structure determination of ten ginsenoside standards. Negative ion experiments made possible the determination of the molecular mass of each ginsenoside standard, the mass of the triterpene core and the masses and sequences of the sugar residues. Positive ion ESMS experiments with the alkali metal cations Li+ or Na+ and the transition metal cations Co2+, Ni2+ and Zn2+ were also useful in determining molecular masses. These alkali and transition metal cations form strongly bonded attachment ions with the ginsenosides. As a result, the CID mass spectra of the metal attachment ions show a variety of (structure characteristic) fragmentations. These experiments can be used to determine the identity of the triterpene core, the types and attachment points of sugars to the core and the nature of the O-glycosidic linkages in the appended disaccharides. Combining the results from the negative and positive ion experiments provides a promising approach to the structure analysis of this class of natural products.     

Molecular dynamics calculation of half-lives for thermal decay of Lennard-Jones clusters

Molecular dynamics has been used with a Lennard-Jones (6–12) potential in order to study the decay behavior of neutral Argon clusters containing between 12 and 14 atoms. The clusters were heated to temperatures well above their melting points and then tracked in time via molecular dynamics until evaporation of one or more atoms was observed. In each simulation, the mode of evaporation, energy released during evaporation, and cluster lifetime were recorded. Results from roughly 2000 simulation histories were combined in order to compute statistically significant values of cluster half-lives and decay energies. It was found that cluster half-life decreases with increasing energy and that for a given value of excess energy (defined asE=(E _tot ?E _gnd)/n), the 13 atom cluster is more stable against decay than clusters containing either 12 or 14 atoms. The dominant decay mechanism for all clusters was determined to be single atom emission.     

On the structure of CH5+; A study of hydron transfer reactions from CH4H+ and CD4H+ tandem-ICR

Relative rates of proton and deuteron transfer from CH₄D⁺ and CD₄H⁺ to a number of molecules were examined in a tandem-ion cyclotron resonance instrument. The results were in conflict with the recent work of Sefcik et al. and support a randomized model.     

Urinary arsenic speciation by high-performance liquid chromatography/atomic absorption spectrometry for monitoring occupational exposure to inorganic arsenic

Total urinary arsenic determinations are often used to assess occupational exposure to inorganic arsenic. Ingestion of sea food can increase the normal background levels of total arsenic in urine by up to an order of magnitude, but this arsenic has relatively little toxicity; it is tightly bound as arsenobetaine. The excretion of inorganic arsenic and its metabolites dimethylarsenic acid (DMA) and monomethylarsonic acid (MMA) is not influenced by the consumption of arsenic from sea food. Specific measurements of DMA, MMA and inorganic arsenic provide a more reliable indicator or exposure than total urinary arsenic levels. An automated atomic absorption method involving high-performance liquid chromatographic separation of the arsenic species and continuous hydride generation is described for the determination of arsenite, arsenate, DMA and MMA at μg As l^?1 levels. The method is used to study normal urinary arsenic levels in laboratory staff and arsenic excretion by exposed workers.     

Proteome analyses using accurate mass and elution time peptide tags with capillary LC time-of-flight mass spectrometry

We describe the application of capillary liquid chromatography (LC) time-of-flight (TOF) mass spectrometric instrumentation for the rapid characterization of microbial proteomes. Previously (Lipton et al., Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 11049) the peptides from a series of growth conditions of Deinococcus radiodurans have been characterized using capillary LC MS/MS and accurate mass measurements which are captured as an accurate mass and time (AMT) tag database. Using this AMT tag database, detected peptides can be assigned using measurements obtained on a TOF due to the additional use of elution time data as a constraint. When peptide matches are obtained using AMT tags (i.e., using both constraints) unique matches of a mass spectral peak occurs 88% of the time. Not only are AMT tag matches unique in most cases, the coverage of the proteome is high; approximately 3500 unique peptide AMT tags are found on average per capillary LC run. From the results of the AMT tag database search, approximately 900 ORFs detected using LC-TOFMS, with approximately 500 ORFs covered by at least two AMT tags. These results indicate that AMT database searches with modest mass and elution time criteria can provide proteomic information for approximately one thousand proteins in a single run of <3 h. The advantage of this method over using MS/MS based techniques is the large number of identifications that occur in a single experiment as well as the basis for improved quantitation. For MS/MS experiments, the number of peptide identifications is severely restricted because of the time required to dissociate the peptides individually. These results demonstrate the utility of the AMT tag approach using capillary LC-TOF MS instruments, and also show that AMT tags developed using other instrumentation can be effectively utilized.     

Rydberg states in fluorinated benzenes; hexa-, penta-, and monofluorobenzene

Spectra of fluorobenzene (FB), pentafluorobenzene (PFB) and hexafluorobenzene (HFB) from 50000 to 93000 cm⁻¹ are presented. Rydberg series converging to the first and to a higher ionization limit are found in each molecule. The orbital natures of the excited states and ions are discussed.     

Lipophilicity in PK design: methyl, ethyl, futile

Lipophilicity, often expressed as distribution coefficients (log D) in octanol/water, is an important physicochemical parameter influencing processes such as oral absorption, brain uptake and various pharmacokinetic (PK) properties. Increasing log D values increases oral absorption, plasma protein binding and volume of distribution. However, more lipophilic compounds also become more vulnerable to P450 metabolism, leading to higher clearance. Molecular size and hydrogen bonding capacity are two other properties often considered as important for membrane permeation and pharmacokinetics. Interrelationships among these physicochemical properties are discussed. Increasing size (molecular weight) often gives higher potency, but inevitably also leads to either higher lipophilicity, and hence poorer dissolution/solubility, or to more hydrogen bonding capacity, which limits oral absorption. Differences in optimal properties between gastrointestinal absorption and uptake into the brain are addressed. Special attention is given to the desired lipophilicity of CNS drugs. In examples using -blockers, Ca channel antagonists and peptidic renin inhibitors we will demonstrate how potency and pharmacokinetic properties need to be balanced.