Marcel Asso

Isolation and characterization of the pyruvate-ferredoxin oxidoreductase from the sulfate-reducing bacterium Desulfovibrio africanus

We report the first purification and characterization of a pyruvate-ferredoxin oxidoreductase (POR) from a sulfate-reducing bacterium, Desulfovibrio africanus. The enzyme as isolated is highly stable in the presence of oxygen and exhibits a specific activity of 14 U/mg. D. africanus POR is a 256 kDa homodimer which contains thiamine pyrophosphate (TPP) and iron-sulfur clusters. EPR spectroscopic study of the enzyme indicates the presence of three [4Fe-4S]2+/1- centers/subunits. The midpoint potentials of the three centers are -390 mV, -515 mV and -540 mV. The catalytic mechanism of POR involves a free radical intermediate which disappears when coenzyme A is added. This behaviour is discussed in terms of an electron-transport chain from TPP to the acceptor. The enzyme activated by dithioerythritol shows an exceptionally high activity compared with other mesophile PORs and becomes very sensitive to oxygen in contrast to the enzyme before activation. The comparison of EPR spectra given by the as isolated and activated enzymes shows that neither the nature, nor the arrangement of FeS centers are affected by the activation process. D. africanus ferredoxins I and II are involved as the physiological electron carriers of the enzyme. POR was shown to be located in the cytoplasm by immunogold labelling.

EPR spectroscopy: A powerful technique for the structural and functional investigation of metalloproteins

Numerous metal centers in proteins can be prepared in a redox state in which their ground state is paramagnetic. Complementary data provided by EPR, Mössbauer, electron nuclear double resonance, magnetic circular dichroism, and NMR spectroscopies have therefore played a major role in the elucidation of the structure and function of these centers. Among those techniques the most commonly used is certainly EPR spectroscopy. In this article various aspects of the current applications of EPR to the structural and functional study of metalloproteins are presented. They are illustrated by recent studies carried out in our laboratory in the field of metalloenzymes and electron transfer systems. The power of numerical simulation techniques is emphasized throughout this work.

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 (0.8–100 cm−1) for the parameter J0 of the isotropic interaction J0SA.SB, but neither the magnitude of J0 nor its sign could be further specifie...

Is there a rate-limiting step in the catalytic cycle of [Ni-Fe] hydrogenases?

Abstract. The question of the existence of a rate-limiting step in the catalytic cycle of Ni-Fe hydrogenases was taken up by using the sets of data available in the case of two specific enzymes: the hydrogenase from Thiocapsa roseopercisina, in which isotope effects have been systematically investigated over a wide pH range, and the enzyme from Desulfovibrio fructosovorans, for which the activities and the redox properties have been studied in two different forms, the wild type and the P238C mutant. When these data are analyzed in the light of appropriate kinetic models, it is concluded that electron transfer and proton transfer are rate limiting in the H2 uptake and H2 evolution reactions, respectively. This proposal is consistent with the data available from other Ni-Fe enzymes.

A new approach for the structural study of metalloproteins: the quantitative analysis of intercenter magnetic interactions

Abstract The quantitative analysis of intercenter magnetic interactions, based on the simulation of EPR spectra recorded at different microwave frequencies, is a powerful technique to determine the relative arrangement of paramagnetic centers in metalloproteins. Such simulations generally rely on a model Hamiltonian in which the interacting centers are approximated by point dipoles. This approximation is often sufficient when these centers are mononuclear metal complexes or organic radicals in which the spin density is not too delocalized and keeps a constant sign. It is used in the present paper to study the magnetic interactions among several hemes and between a heme and a FMN radical in cytochromes. In the case of metalloproteins containing polynuclear metal clusters, the point dipole approximation is no longer valid and must be replaced by a local spin model in which the magnetic interactions among all the paramagnetic sites of the system are explicitly considered. Numerical simulations based on this model provide a description of the relative arrangement of the interacting centers at atomic resolution and can be used to assign a valence state to the different metal ions of the clusters. This is illustrated by recent studies carried out on metalloproteins containing [2Fe-2S]1+ and [4Fe-4S]1+ clusters.

Peptide-lanthanide cation equilibria in aqueous phase. I. Bound shifts for l-carnosine-praseodymium complexes

Abstract l -Carnosine complexes of Pr3+ were characterized in aqueous solution by 1H NMR and potentiometric titration. A rigorous treatment of chemical shifts and pH variation data with lanthanide concentration is presented. Two different forms of the peptide ligand, forming simultaneously two complexes, were taken into account. At low pH values the cation is only coordinated at the carboxylate site of the ligand in a weak complex (β2 = 6) whereas in neutral solution a stronger complex (β1 = 37) is present as a consequence of the deprotonation of the imidazole ring. The computation of induced bound shifts † 2 and Δ1 for resonating nuclei of the peptide in both forms yields consistent figures. These provide the experimental basis for a conformational model which is usually not obtainable for labile complexes with low stability constants.

Individual redox characteristics and kinetic properties of the hemes in cytochromes c3: New methods of investigation

The elucidation of the role of the four hemes in cytochromes c3 requires several complementary approaches. The measurements and the assignment of the redox potentials resort to magnetic spectroscopies, EPR and NMR, which are able to discriminate the hemes. The origin of the differences between the redox properties of the hemes can be studied by comparing their thermodynamic parameters delta S and delta H, as measured by the temperature dependence of their individual potentials. Lastly, the available data concerning the electron exchange between cytochromes c3 and their redox partners can be analysed through a detailed kinetic model which provides important information on the role of the different hemes.

Cytochrome c3-ferredoxin I covalent complex: evidence for an intramolecular electron exchange in cytochrome c3

Desulfovibrio desulfuricans Norway tetraheme cytochrome c3 and (4Fe-4S) cluster containing ferredoxin I are physiological electron-transfer partners. To obtain structural informations on the complex formation, a covalent cytochrome c3-ferredoxin I complex was used. EPR investigations suggest that the covalent cytochrome c3-ferredoxin I complex is a stable analogue of the native one and bring experimental support to an intramolecular electron exchange in cytochrome c3. The reduction of cross-linked ferredoxin I under a hydrogen atmosphere in the presence of hydrogenase necessitates an intramolecular electron exchange between the hemes of cytochrome c3. A spectrophotometric redox titration indicates that the redox potentials of cytochrome c3 are weakly affected by the complex formation.

Zinc (II) and Nickel (II) Complexes of Cysteinyl-Cysteine

Abstract Complexes of transition metals with cysteinyl-cysteine: cys-cys are of interest as models for metalloproteins such as metallothionein. Infrared and Raman Spectra of cys-cys, Ni (II)- (cys-cys)2 and Zn (II)-(cys-cys)2 complexes are reported. The results show that the dipeptide is bound to transition metals through sulfur only.

Peptide-lanthanide cation equilibria in aqueous solution. II. Isomorphous replacement and separation of the contact and pseudo-contact contributions to NMR shifts induced by a paramagnetic ion

Abstract Labile complexes of Pr, Nd, Eu, Dy, Ho, and Yb cations with L -camosine (β-ala-nylhistidine) have been investigated by NMR in aqueous acidic solution. Paramagnetic bound shifts (Δ2) values for all proton resonances have been deduced and in the case of CH2N and CH2C protons where no contact shift is observed, isomorphous replacement and effective axial symmetry is confirmed for the Ln3+ series. For contact shifted nuclei, the separation of contact and pseudo-contact terms has been effected by the use of computed Δ2 values. From these results it appears that the hyperfine coupling constant (Ai) cannot be kept identical throughout the lanthanide elements for isostructural complexes.