Olivier Hamelin

Involvement of mitochondrial ferredoxin and para-aminobenzoic acid in yeast coenzyme Q biosynthesis.

Yeast ubiquinone or coenzyme Q(6) (Q(6)) is a redox active lipid that plays a crucial role in the mitochondrial electron transport chain. At least nine proteins (Coq1p-9p) participate in Q(6) biosynthesis from 4-hydroxybenzoate (4-HB). We now show that the mitochondrial ferredoxin Yah1p and the ferredoxin reductase Arh1p are required for Q(6) biosynthesis, probably for the first hydroxylation of the pathway. Conditional Gal-YAH1 and Gal-ARH1 mutants accumulate 3-hexaprenyl-4-hydroxyphenol and 3-hexaprenyl-4-aminophenol. Para-aminobenzoic acid (pABA) is shown to be the precursor of 3-hexaprenyl-4-aminophenol and to compete with 4-HB for the prenylation reaction catalyzed by Coq2p. Yeast cells convert U-((13)C)-pABA into (13)C ring-labeled Q(6), a result that identifies pABA as a new precursor of Q(6) and implies an additional NH(2)-to-OH conversion in Q(6) biosynthesis. Our study identifies pABA, Yah1p, and Arh1p as three actors in Q(6) biosynthesis.

Photocatalyzed sulfide oxygenation with water as the unique oxygen atom source.

In our research program aiming to develop new ruthenium-based polypyridine catalysts for oxidation we were interested in combining a photosensitizer and a catalytic fragment within the same complex to achieve catalytic light-driven oxidation. To respond to the lack of such conjugates, we report here a new catalytic system capable of using light to activate water molecules in order to perform selective sulfide oxygenation into sulfoxide via an oxygen atom transfer from H(2)O to the substrate with a TON of up to 197 ± 6. On the basis of electrochemical and photophysical studies, a proton-coupled electron-transfer process yielding to an oxidant Ru(IV)-oxo species was proposed. In particular, the synergistic effect between both partners in the dyad yielding a more efficient catalyst compared to the bimolecular system is highlighted.

A Dyad as Photocatalyst for Light-Driven Sulfide Oxygenation with Water As the Unique Oxygen Atom Source

With the objective to convert light energy into chemical oxidation energy, a ruthenium-based dyad constituted of the assembly of a photosensitizer and a catalytic fragment was synthesized. Upon irradiation with blue LEDs, and in the presence of an electron acceptor, the complex is able to catalyze selective sulfide oxygenation involving an oxygen atom transfer from water to the substrate. Electrochemical and photophysical studies highlighted a proton-coupled electron transfer (PCET) to access to a high valent oxidant Ru(IV) oxo species.

ubiI, a new gene in Escherichia coli coenzyme Q biosynthesis, is involved in aerobic C5-hydroxylation.

Background: The C5-hydroxylation reaction of coenzyme Q biosynthesis in Escherichia coli is catalyzed by an unknown enzyme. Results: The UbiI protein is responsible for the C5-hydroxylation reaction. Conclusion: The three monooxygenases involved in aerobic Q biosynthesis are now identified. Significance: We report the characterization of a gene of unknown function and the first crystal structure of a monooxygenase involved in Q biosynthesis. Coenzyme Q (ubiquinone or Q) is a redox-active lipid found in organisms ranging from bacteria to mammals in which it plays a crucial role in energy-generating processes. Q biosynthesis is a complex pathway that involves multiple proteins. In this work, we show that the uncharacterized conserved visC gene is involved in Q biosynthesis in Escherichia coli, and we have renamed it ubiI. Based on genetic and biochemical experiments, we establish that the UbiI protein functions in the C5-hydroxylation reaction. A strain deficient in ubiI has a low level of Q and accumulates a compound derived from the Q biosynthetic pathway, which we purified and characterized. We also demonstrate that UbiI is only implicated in aerobic Q biosynthesis and that an alternative enzyme catalyzes the C5-hydroxylation reaction in the absence of oxygen. We have solved the crystal structure of a truncated form of UbiI. This structure shares many features with the canonical FAD-dependent para-hydroxybenzoate hydroxylase and represents the first structural characterization of a monooxygenase involved in Q biosynthesis. Site-directed mutagenesis confirms that residues of the flavin binding pocket of UbiI are important for activity. With our identification of UbiI, the three monooxygenases necessary for aerobic Q biosynthesis in E. coli are known.

A Ruthenium(II)–Copper(II) Dyad for the Photocatalytic Oxygenation of Organic Substrates Mediated by Dioxygen Activation

Dioxygen activation by copper complexes is a valuable method to achieve oxidation reactions for sustainable chemistry. The development of a catalytic system requires regeneration of the Cu(I) active redox state from Cu(II). This is usually achieved using extra reducers that can compete with the Cu(II)(O2) oxidizing species, causing a loss of efficiency. An alternative would consist of using a photosensitizer to control the reduction process. Association of a Ru(II) photosensitizing subunit with a Cu(II) pre-catalytic moiety assembled within a unique entity is shown to fulfill these requirements. In presence of a sacrificial electron donor and light, electron transfer occurs from the Ru(II) center to Cu(II). In presence of dioxygen, this dyad proved to be efficient for sulfide, phosphine, and alkene catalytic oxygenation. Mechanistic investigations gave evidence about a predominant (3)O2 activation pathway by the Cu(I) moiety.

Multistep anchoring of a catalytically active ruthenium complex in porous mesostructured silica

A supported [Ru(dmp)2(Py@LUS)Cl]Cl complex (dmp = 2,9-dimethyl-1,10-phenantroline) was synthesised in LUS, a 2D hexagonal porous mesostructured silica, via a step-by-step approach for the sake of site isolation, unicity and localisation in the confined space of the nanopores of the silica matrix. A pyridine terminated tether, Py@LUS, was homogeneously distributed on the surface using a molecular stencil patterning technique, followed by reaction of [Ru(dmp)2Cl2] at 78 °C. All intermediate materials were thoroughly characterised with a panel of techniques, including X-ray diffraction, elemental analysis, 29Si and 13C solid state NMR, diffuse reflectance UV-vis and FT-IR spectroscopies, and cyclovoltamperometry. Site isolation, unicity and localisation are achieved in the confined space of the nanopores of the silica matrix. The final material is selectively active in the catalytic oxidation of methylphenylsulfide into sulfoxide.

Studies of Inhibitor Binding to the [4Fe-4S] Cluster of Quinolinate Synthase†

Stop for NadA! A [4Fe-4S] enzyme, NadA, catalyzes the formation of quinolinic acid in de novo nicotinamide adenine dinucleotide (NAD) biosynthesis. A structural analogue of an intermediate, 4,5-dithiohydroxyphthalic acid (DTHPA), has an in vivo NAD biosynthesis inhibiting activity in E. coli. The inhibitory effect can be explained by the coordination of DTHPA thiolate groups to a unique Fe site of the NadA [4Fe-4S] cluster.

A simple biosynthetic method for stereospecific resonance assignment of prochiral methyl groups in proteins

A new method for stereospecific assignment of prochiral methyl groups in proteins is presented in which protein samples are produced using U-[13C]glucose and subsaturating amounts of 2-[13C]methyl-acetolactate. The resulting non-uniform labeling pattern allows proR and proS methyl groups to be easily distinguished by their different phases in a constant-time two-dimensional 1H-13C correlation spectra. Protein samples are conveniently prepared using the same media composition as the main uniformly-labeled sample and contain higher levels of isotope-enrichment than fractional labeling approaches. This new strategy thus represents an economically-attractive, robust alternative for obtaining isotopically-encoded stereospecific NMR assignments of prochiral methyl groups.

CH3-specific NMR assignment of alanine, isoleucine, leucine and valine methyl groups in high molecular weight proteins using a single sample

Abstract A new strategy for the NMR assignment of aliphatic side-chains in large perdeuterated proteins is proposed. It involves an alternative isotopic labeling protocol, the use of an out-and-back 13C–13C TOCSY experiment ((H)C-TOCSY-C-TOCSY-(C)H) and an optimized non-uniform sampling protocol. It has long been known that the non-linearity of an aliphatic spin-system (for example Ile, Val, or Leu) substantially compromises the efficiency of the TOCSY transfers. To permit the use of this efficient pulse scheme, a series of optimized precursors were designed to yield linear 13C perdeuterated side-chains with a single protonated CH3 group in these three residues. These precursors were added to the culture medium for incorporation into expressed proteins. For Val and Leu residues, the topologically different spin-systems introduced for the pro-R and pro-S methyl groups enable stereospecific assignment. All CH3 can be simultaneously assigned on a single sample using a TOCSY experiment. It only requires the tuning of a mixing delay and is thus more versatile than the relayed COSY experiment. Enhanced resolution and sensi-tivity can be achieved by non-uniform sampling combined with the removal of the large JCC coupling by deconvolution prior to the processing by iterative soft thresholding. This strategy has been used on malate synthase G where a large percentage of the CH3 groups could be correlated directly up to the backbone Ca. It is anticipated that this robust combined strategy can be routinely applied to large proteins.

Efficient, Highly Stereocontrolled First Synthesis of (-)-9-Acetoxyfukinanolide from Enantiopure 1,6-Dimethylcyclohexene

Abstract A particularly efficacious first preparation of enantiopure (-)-9-acetoxyfukinanolide has been carried out that is based on a stereoselective dichloroketene-1, 6-dimethylcyclohexene cycloaddition reaction and effective free-radical β-methylene-γ-butyrolactonization and retroaldol-aldol processes.