Chemical segregation of complex organic O-bearing species in Orion KL.

We investigate the chemical segregation of complex O-bearing species (including the largest and most complex ones detected to date in space) towards Orion KL, the closest high-mass star-forming region. The molecular line images obtained using the ALMA science verification data reveal a clear segregation of chemically related species depending on their different functional groups. We map the emission of 13CH3OH, HCOOCH3, CH3OCH3, CH2OCH2, CH3COOCH3, HCOOCH2CH3, CH3CH2OCH3, HCOOH, OHCH2CH2OH, CH3COOH, CH3CH2OH, CH3OCH2OH, OHCH2CHO, and CH3COCH3 with ~1.5″ angular resolution and provide molecular abundances of these species toward different gas components of this region. We disentangle the emission of these species in the different Orion components by carefully selecting lines free of blending and opacity effects. Possible effects in the molecular spatial distribution due to residual blendings and different excitation conditions are also addressed. We find that while species containing the C-O-C group, i.e. an ether group, exhibit their peak emission and higher abundance towards the compact ridge, the hot core south is the component where species containing a hydroxyl group (-OH) bound to a carbon atom (C-O-H) present their emission peak and higher abundance. This finding allows us to propose methoxy (CH3O-) and hydroxymethyl (-CH2OH) radicals as the major drivers of the chemistry in the compact ridge and the hot core south, respectively, as well as different evolutionary stages and prevailing physical processes in the different Orion components.

Searching for trans ethyl methyl ether in Orion KL.

We report on the tentative detection of trans ethyl methyl ether (tEME), t-CH3CH2OCH3, through the identification of a large number of rotational lines from each one of the spin states of the molecule towards Orion KL. We also search for gauche-trans-n-propanol, Gt-n-CH3CH2CH2OH, an isomer of tEME in the same source. We have identified lines of both species in the IRAM 30 m line survey and in the ALMA Science Verification data. We have obtained ALMA maps to establish the spatial distribution of these species. Whereas tEME mainly arises from the compact ridge component of Orion, Gt-n-propanol appears at the emission peak of ethanol (south hot core). The derived column densities of these species at the location of their emission peaks are ≤(4.0 ± 0.8) × 1015 cm-2 and ≤(1.0 ± 0.2)× 1015 cm-2 for tEME and Gt-n-propanol, respectively. The rotational temperature is ~100 K for both molecules. We also provide maps of CH3OCOH, CH3CH2OCOH, CH3OCH3, CH3OH, and CH3CH2OH to compare the distribution of these organic saturated O-bearing species containing methyl and ethyl groups in this region. Abundance ratios of related species and upper limits to the abundances of non-detected ethers are provided. We derive an abundance ratio N(CH3OCH3)/N(tEME) ≥ 150 in the compact ridge of Orion.

Studies on protoporphyrin biosynthetic pathway in Saccharomyces cerevisiae ; characterization of the tetrapyrrole intermediates.

An acellular extract of the yeast, Saccharomyces cerevisiae, incubated with ALA, is able to synthesize protoporphyrin from this precursor. Several tetrapyrrole intermediates were extracted from the medium and purified by silica gel chromatography. The chromatographic behaviour and the spectral properties of the isolated seven free carboxylic porphyrins (and of the corresponding esters), show that each product has a different carboxyle number, varying from eight (uroporphyrin) to two (protoporphyrin). The identification of five of them (octa- to tetracarboxymethyl-porphyrinester) is confirmed by mass spectrometry. The effect of physical factors (temperature, pH, time) on the protoporphyrin biosynthesis system indicates that the enzymes catalysing the first steps of the pathway (ALA leads to Coproporphyrin) are more stable than those catalysing the last steps (Coproporphyrin leads to Protoporphyrin). Results obtained with some enzymatic inhibitors (EDTA, OP, pCMB) show the sensitivity of the ALA dehydratase to OP and to pCMB (confirming therefore its nature as a metallo- and sulfhydryl enzyme) and also of the overall porphyrin synthesis system to these three agents.

Purification and properties of uroporphyrinogen decarboxylase from Saccharomyces cerevisiae. Yeast uroporphyrinogen decarboxylase.

Uroporphyrinogen decarboxylase (EC 4.1.1.37) was purified about 14000-fold to homogeneity from the yeast Saccharomyces cerevisiae with a 70% overall yield. The purification included affinity chromatography on uroporphyrin-I-Affi-Gel 102. The specific activity of the final preparation was 1750 nmol coproporphyrinogen formed.h-1.(mg protein)-1 at pH 7.5 and 37 degrees C using 4 microM uroporphyrinogen I as substrate. The purified enzyme has a minimum molecular mass of 38 kDa by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and 46 kDa by gel filtration, suggesting that yeast uroporphyrinogen decarboxylase is a monomer. Chromatofocusing gave a pI of 6.0. Enzyme activity was inhibited by metals, such as Cu2+, Zn2+, Fe2+, Fe3+ and by sulfhydryl-specific reagents, but no cofactor requirement could be demonstrated. The optimum pH was pH 5.7 for uroporphyrinogens I and III and heptacarboxylate porphyrinogen I as estimated by coproporphyrinogen formation. The optimum pH for substrate decarboxylation was pH 5.7 for uroporphyrinogen I, but pH 6.8 for the two other substrates. The Km values at pH 5.7 were 10 nM for uroporphyrinogen I, 6 nM for uroporphyrinogen III and 7 nM for heptacarboxylate porphyrinogen I as measured by coproporphyrinogen formation. The pattern of accumulation of intermediate and final decarboxylation products and the rates of the successive decarboxylations were determined for the three substrates at different concentrations at pH 5.7 and pH 6.8. The rate-limiting step at 4 microM substrate concentration was the elimination of the second carboxyl group of uroporphyrinogen III and the fourth carboxyl of uroporphyrinogen I. An antiserum to purified yeast uroporphyrinogen decarboxylase was used to characterize the protein in several mutants.

Purification and properties of protoporphyrinogen oxidase from the yeast Saccharomyces cerevisiae. Mitochondrial location and evidence for a precursor form of the protein.

Protoporphyrinogen oxidase, the molecular target of diphenylether-type herbicides, was purified to homogeneity from yeast mitochondrial membranes and found to be a 55-kDa polypeptide with a pI of 8.5 and a specific activity of 40,000 nmol of protoporphyrin/h/mg of protein at 30 degrees C. The Michaelis constant (Km) for protoporphyrinogen IX was 0.1 microM. Due to the high affinity of the enzyme toward oxygen, the Km for oxygen could only be approximated to 0.5-1.5 microM. The purified enzyme contained a flavin as cofactor. Studies with rabbit antibodies to yeast protoporphyrinogen oxidase showed that the enzyme is synthesized as a high molecular weight precursor (58 kDa) that is rapidly converted in vivo to the mature (55 kDa) membrane-bound form. Protoporphyrinogen oxidase activity was found only in purified yeast mitochondrial inner membrane (not in the outer membrane). Acifluorfen-methyl, a potent diphenylether-type herbicide, competitively inhibited the purified enzyme (Ki = 10 nM). The mixed inhibition by acifluorfen-methyl previously reported for the membrane-bound protoporphyrinogen oxidase (Camadro, J.M., Matringe, M., Scalla, R., and Labbe, P. (1991) Biochem. J. 277, 17-21) was shown to be related to partial proteolysis of the enzyme.

Photoaffinity labeling of protoporphyrinogen oxidase, the molecular target of diphenylether-type herbicides.

Diphenylether-type herbicides are extremely potent inhibitors of protoporphyrinogen oxidase, a membrane-bound enzyme involved in the heme and chlorophyll biosynthesis pathways. Tritiated acifluorfen and a diazoketone derivative of tritiated acifluorfen were specifically bound to a single class of high-affinity binding sites on yeast mitochondrial membranes with apparent dissociation constants of 7 nM and 12.5 nM, respectively. The maximum density of specific binding sites, determined by Scatchard analysis, was 3 pmol.mg-1 protein. Protoporphyrinogen oxidase specific activity was estimated to be 2500 nmol protoporphyrinogen oxidized h-1.mol-1 enzyme. The diazoketone derivative of tritiated acifluorfen was used to specifically photolabel yeast protoporphyrinogen oxidase. The specifically labeled polypeptide in wild-type mitochondrial membranes had an apparent molecular mass of 55 kDa, identical to the molecular mass of the purified enzyme. This photolabeled polypeptide was not detected in a protoporphyrinogen-oxidase-deficient yeast strain, but the membranes contained an equivalent amount of inactive immunoreactive protoporphyrinogen oxidase protein.