Serge Gambarelli

Activation of the anaerobic ribonucleotide reductase by S-adenosylmethionine.

In all living organisms, deoxyribonucleotides, the precursors of DNA, are produced by reduction of the corresponding ribonucleotides. The reaction is catalyzed by an enzyme called ribonucleotide reductase (RNR), which is thus absolutely essential for growth and survival. A number of facultative and strict anaerobes depend on a class III RNR, which is characterized by the presence of a catalytically essential and oxygen-sensitive glycyl radical in the active site. The introduction of the radical into the RNR protein is initiated by a second protein (the activase), which is a member of the recently discovered “radicalSAM” enzyme superfamily. The enzymes of the “radical-SAM” family are characterized by a (4Fe 4S) center, which is chelated by the three cysteines of the conserved Cys-X3-Cys-X2-Cys motif and serving for binding, reducing, and cleaving S-adenosylmethionine (SAM) into methionine and a putative 5’-deoxyadenosyl radical (Ado). It is now generally accepted that, in all these systems, a cluster–SAM complex is formed as a reaction intermediate, since such a complex has been directly observed by ENDOR spectroscopy in the cases of pyruvate-formate lyase activase (PFL) and lysine aminomutase (LAM) and by X-ray crystallography in the cases of biotin synthase (BioB), coproporphyrinogen oxidase (HemN), and MoaA, an enzyme involved in the biosynthesis of the Mo cofactor. It is likely that a cluster–SAM complex is also generated in the activase of the RNR as a precursor of the Ado radical. Glycyl radical formation implies radical transfer from one protein (activase) to the other (RNR). In this work, we investigate the question of whether this transfer occurs by direct attack of Ado8 onto the glycyl residue of the RNR active site or through radical relays along a radical-transfer chain connecting the activase to the RNR. For this purpose, we used HYSCORE (Hyperfine Sublevel Correlation) spectroscopy to demonstrate the intermediate formation of a cluster–SAM complex in the activase and label-transfer experiments with RNR preparations

tRNA-modifying MiaE protein from Salmonella typhimurium is a nonheme diiron monooxygenase

MiaE catalyzes the posttranscriptional allylic hydroxylation of 2-methylthio-N-6-isopentenyl adenosine in tRNAs. The Salmonella typhimurium enzyme was heterologously expressed in Escherichia coli. The purified enzyme is a monomer with two iron atoms and displays activity in in vitro assays. The type and properties of the iron center were investigated by using a combination of UV-visible absorption, EPR, HYSCORE, and Mössbauer spectroscopies which demonstrated that the MiaE enzyme contains a nonheme dinuclear iron cluster, similar to that found in the hydroxylase component of methane monooxygenase. This is the first example of an enzyme from this important class of diiron monooxygenases to be involved in the hydroxylation of a biological macromolecule and the second example of a redox metalloenzyme participating in tRNA modification.

Optimization of an absolute sensitivity in a glassy matrix during DNP-enhanced multidimensional solid-state NMR experiments.

Thanks to instrumental and theoretical development, notably the access to high-power and high-frequency microwave sources, high-field dynamic nuclear polarization (DNP) on solid-state NMR currently appears as a promising solution to enhance nuclear magnetization in many different types of systems. In magic-angle-spinning DNP experiments, systems of interest are usually dissolved or suspended in glass-forming matrices doped with polarizing agents and measured at low temperature (down to ∼100K). In this work, we discuss the influence of sample conditions (radical concentration, sample temperature, etc.) on DNP enhancements and various nuclear relaxation times which affect the absolute sensitivity of DNP spectra, especially in multidimensional experiments. Furthermore, DNP-enhanced solid-state NMR experiments performed at 9.4 T are complemented by high-field CW EPR measurements performed at the same magnetic field. Microwave absorption by the DNP glassy matrix is observed even below the glass transition temperature caused by softening of the glass. Shortening of electron relaxation times due to glass softening and its impact in terms of DNP sensitivity is discussed.

An EPR/HYSCORE, Mössbauer, and resonance Raman study of the hydrogenase maturation enzyme HydF: a model for N-coordination to [4Fe–4S] clusters

The biosynthesis of the organometallic H cluster of [Fe–Fe]xa0hydrogenase requires three accessory proteins, two of which (HydE and HydG) belong to the radical S-adenosylmethionine enzyme superfamily. The third, HydF, is an Fe–S protein with GTPase activity. The [4Fe–4S] cluster of HydF is bound to the polypeptide chain through only the three, conserved, cysteine residues present in the binding sequence motif CysXHisX(46-53)HisCysXXCys. However, the involvement of the two highly conserved histidines as a fourth ligand for the cluster coordination is controversial. In this study, we set out to characterize further the [4Fe–4S] cluster of HydF using Mössbauer, EPR, hyperfine sublevel correlation (HYSCORE), and resonance Raman spectroscopy in order to investigate the influence of nitrogen ligands on the spectroscopic properties of [4Fe–4S]2+/+ clusters. Our results show that Mössbauer, resonance Raman, and EPR spectroscopy are not able to readily discriminate between the imidazole-coordinated [4Fe–4S] cluster and the non-imidazole-bound [4Fe–4S] cluster with an exchangeable fourth ligand that is present in wild-type HydF. HYSCORE spectroscopy, on the other hand, detects the presence of an imidazole/histidine ligand on the cluster on the basis of the appearance of a specific spectral pattern in the strongly coupled region, with a coupling constant of approximately 6xa0MHz. We also discovered that a His-tagged version of HydF, with a hexahistidine tag at the N-terminus, has a [4Fe–4S] cluster coordinated by one histidine from the tag. This observation strongly indicates that care has to be taken in the analysis of data obtained on tagged forms of metalloproteins.

Magnetic interactions between a [4Fe–4S]1+ cluster and a flavin mononucleotide radical in the enzyme trimethylamine dehydrogenase: A high-field electron paramagnetic resonance study

Trimethylamine dehydrogenase is a bacterial enzyme which contains two redox centers: a flavin mononucleotide (FMN) group which constitutes the active site and a [4Fe–4S]1+,2+ cluster which transfers the electrons provided by the FMN to an electron-transferring flavoprotein. According to the x-ray crystal structure, the center-to-center distance is equal to 12 A and the nearest atoms of the two centers are separated by a 4 A gap. Although this arrangement does not appear especially favorable for mediating strong magnetic interactions, a triplet state electron paramagnetic resonance (EPR) spectrum arising from the intercenter magnetic coupling is observed at X band (9 GHz) when the enzyme is reduced by its substrate. In earlier work, the temperature dependence of this spectrum and its analysis based on a triplet state spin Hamiltonian were used to propose the range (0.8–100u2009cm−1) for the parameter J0 of the isotropic interaction J0SA.SB, but neither the magnitude of J0 nor its sign could be further specifie...

Investigation of magnetic anisotropy of (Ge,Mn) nanocolumns

Thin films of Ge1−xMnx containing Mn-rich nanostructures, so called nanocolumns, are grown by molecular-beam-epitaxy at low growth temperature (Tg) on Ge(001) substrate. Depending on the growth temperature, these nanocolumns can be either crystalline or amorphous. A quantitative study of magnetic anisotropy of these nanocolumns is performed by superconducting quantum interference device and electron paramagnetic resonance. We present a correlation between the structural morphology (diamond lattice) and the magnetic properties in these nanocolumns. Crystalline (Ge,Mn) nanocolumns (Tg=100u2009°C) exhibit fourth-order magnetic anisotropy due to the coupling between their magnetization and the cubic lattice whereas amorphous columns (Tg=150u2009°C) only exhibit second-order shape anisotropy.

Spin transport in p-type germanium

We report on the spin transport properties in p-doped germanium (Ge-p) using low temperature magnetoresistance measurements, electrical spin injection from a ferromagnetic metal and the spin pumping-inverse spin Hall effect method. Electrical spin injection is carried out using three-terminal measurements and the Hanle effect. In the 2-20 K temperature range, weak antilocalization and the Hanle effect provide the same spin lifetime in the germanium valence band (≈1 ps) in agreement with predicted values and previous optical measurements. These results, combined with dynamical spin injection by spin pumping and the inverse spin Hall effect, demonstrate successful spin accumulation in Ge. We also estimate the spin Hall angle θ(SHE) in Ge-p (6-7 x 10(-4) at room temperature, pointing out the essential role of ionized impurities in spin dependent scattering.

Structure and magnetism of Ge3Mn5 clusters

We have grown Ge3Mn5 clusters by codepositing germanium and manganese atoms on Ge(001) substrates using low temperature molecular beam epitaxy and further annealing the films at high temperature. Clusters are spherical and randomly distributed in the germanium film in epitaxial relationship with the diamond lattice. They exhibit a broad size distribution. By performing a careful x-ray diffraction analysis, we could find that 97% of Ge3Mn5 clusters have their c-axis perpendicular to the film plane while 3% exhibit in-plane c-axis. We could also show a slight in-plane distortion of the Ge3Mn5 lattice leading to a reduction of uniaxial magnetic anisotropy. These observations are well confirmed by complementary superconducting quantum interference device and electron paramagnetic resonance measurements.

Ferromagnetic spins interaction in alternating branched polyarylamines

The chemical oxidation of alternating branched polyarylamines leads to the formation of radical cations, the presence of which was manifested by the appearance of new bands in the UV–visible–near infrared spectra. Spins of radical cations can be magnetically coupled. The nutation pulsed-EPR technique was used to determine the multiplicity of spin systems for different oxidant:polymer unit ratios. It was found that spins of polymers oxidized to a radical cation in each conjugated amine segment interact magnetically to form dominant quartet state (Su2009=u20093/2). Magnetization measurements confirmed the ferromagnetic interaction of spins, returning an exchange integral Ju2009=u2009+0.75 meV.

An artificial photosynthetic system for photoaccumulation of two electrons on a fused dipyridophenazine (dppz)–pyridoquinolinone ligand

The π-extended ligand of a ruthenium complex stores two photo-generated electrons, mimicking a key step in photosynthesis.