Jacques Gaillard

Purification and properties of a catalase from potato tubers (Solanum tuberosum)

Abstract Catalase from potato tubers (Solanum tuberosum) has been purified to homogeneity. The purified enzyme had a low specific activity (approx. 3000 units/mg of protein) and a marked tendency to form aggregates. Polyacrylamide gel electrophoresis in the presence of SDS revealed a single 56 kDa peptide. The apparent molecular weight of the native protein was estimated to 224 kDa. Some homology was found between the N-terminal sequences of potato, Ipomea batatas, and bovine liver catalases. The enzyme exhibited optical absorbance maxima at 280, 403, 500, 535, and 620 nm, and its epr spectrum was characteristic of a hemoprotein with high-spin ferric iron. The pseudo first order rate constant was invariant for H2O2 concentrations ranging from 8 to 30 mM, but decreased rapidly at higher concentrations. Cyanide and azide were inhibitors, and 2-mercaptoethanol was a much more efficient inhibitor than for other catalases. No NADPH could be detected in the potato catalase; this was the first search for this dinucleotide in a plant catalase and its absence in the potato enzyme could be tentatively related to its lower propensity to form the compound II state.

Characterization of Three XylT-Like [2Fe-2S] Ferredoxins Associated with Catabolism of Cresols or Naphthalene: Evidence for Their Involvement in Catechol Dioxygenase Reactivation

The xylT gene product, a component of the xylene catabolic pathway of Pseudomonas putida mt2, has been recently characterized as a novel [2Fe-2S] ferredoxin which specifically reactivates oxygen-inactivated catechol 2,3-dioxygenase (XylE). In this study, three XylT-like proteins potentially involved in the catabolism of naphthalene (NahT) or cresols (PhhQ and DmpQ) have been overexpressed in Escherichia coli, purified, and compared with respect to their biochemical properties and interaction with XylE. The three XylT analogues show general spectroscopic characteristics common to plant-type [2Fe-2S] ferredoxins as well as distinctive features that appear to be typical for the XylT subgroup of these proteins. The midpoint redox potentials of the PhhQ and DmpQ proteins were -286 mV and -323 mV, respectively. Interestingly, all purified XylT-like proteins promoted in vitro reactivation of XylE almost as efficiently as XylT. The interaction of XylE with XylT and its analogues was studied by cross-linking experiments using the 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. A polypeptide band with an M(r) of 46,000, which corresponded to the cross-linked product between one XylE subunit and one molecule of ferredoxin, was obtained in all cases. The formation of the complex was affected by ionic strength, indicating that electrostatic forces are involved in the dioxygenase-ferredoxin interaction. In complementation experiments, plasmids expressing xylT or its analogues were introduced into an XylT-null mutant of P. putida which is unable to grow on p-methylbenzoate. All transconjugants regained the wild-type phenotype, indicating that all analogues can substitute for XylT in the in vivo reactivation of XylE. Our results provide evidence for a subgroup of [2Fe-2S] ferredoxins with distinct biochemical properties whose specific function is to reactivate intrinsically labile extradiol ring cleavage dioxygenases involved in the catabolism of various aromatic hydrocarbons.

A Second [2Fe-2S] Ferredoxin from Sphingomonas sp. Strain RW1 Can Function as an Electron Donor for the Dioxin Dioxygenase

The first step in the degradation of dibenzofuran and dibenzo-p-dioxin by Sphingomonas sp. strain RW1 is carried out by dioxin dioxygenase (DxnA1A2), a ring-dihydroxylating enzyme. An open reading frame (fdx3) that could potentially specify a new ferredoxin has been identified downstream of dxnA1A2, a two-cistron gene (J. Armengaud, B. Happe, and K. N. Timmis, J. Bacteriol. 180:3954-3966, 1998). In the present study, we report a biochemical analysis of Fdx3 produced in Escherichia coli. This third ferredoxin thus far identified in Sphingomonas sp. strain RW1 contained a putidaredoxin-type [2Fe-2S] cluster which was characterized by UV-visible absorption spectrophotometry and electron paramagnetic resonance spectroscopy. The midpoint redox potential of this ferredoxin (E(0) = -247 +/- 10 mV versus normal hydrogen electrode at pH 8.0) is similar to that exhibited by Fdx1 (-245 mV), a homologous ferredoxin previously characterized in Sphingomonas sp. strain RW1. In in vitro assays, Fdx3 can be reduced by RedA2 (a reductase similar to class I cytochrome P-450 reductases), previously isolated from Sphingomonas sp. strain RW1. RedA2 exhibits a K(m) value of 3.2 +/- 0.3 microM for Fdx3. In vivo coexpression of fdx3 and redA2 with dxnA1A2 confirmed that Fdx3 can serve as an electron donor for the dioxin dioxygenase.

Interaction between pyridine adenine dinucleotides and bovine liver catalase: A chromatographic and spectral study

Two different fractions were present in crystalline bovine liver catalase, and could be resolved using dye-ligand affinity chromatography with Red-A Matrex gel containing Procion HE 3B. The major part (alpha) was not adsorbed on this gel. The second fraction (beta) was firmly adsorbed to the gel, and could be eluted either by high salt or by NADPH in the micromolar range. Elution of catalase beta was also obtained with NADH, NADP+, and ADP at higher concentration. Fractions alpha and beta displayed no detectable difference in specific activity, stability to heat, and light absorption data. It is suggested that the difference in behavior between alpha and beta is related to the binding of NADPH to the mammalian catalase [H. N. Kirkman and G. F. Gaetani (1984) Proc. Natl. Acad. Sci. USA 81, 4343-4347], and that the beta fraction corresponds to the enzyme molecules that have at least one free site for NADPH binding. Modifications of catalase molecules in the presence of dithioerythritol (DTE) were examined using light absorption and EPR data. Thiol induced changes that corresponded to the formation of catalase complex II. They were partially reversed by NADPH at very low level, and the dinucleotide appeared to be oxidized in this process. DTE-treated bovine catalase was totally adsorbed on the Red-A Matrex columns, and could be eluted as fraction beta. Similar spectral changes in the presence of DTE and NADPH were displayed by a bacterial catalase from Proteus mirabilis. This enzyme was also able to oxidize NADPH, but was not adsorbed by Red-A Matrex. This work suggests that dye-affinity chromatography provides a very convenient tool for isolating dinucleotide-depleted catalase from bovine liver, facilitating further study of the physiological function of this cofactor within the enzyme.

Purification and characterization of a 7Fe-ferredoxin from Rhodobacter capsulatus

A ferredoxin was purified anaerobically from Rhodobacter capsulatus grown photoheterotrophically with excess ammonia. This ferredoxin, called ferredoxin II (FdII), had a molecular weight of approximatively 15,000 by gel filtration and 14,000 by SDS polyacrylamide gel electrophoresis indicating that it is monomeric. Its absorption spectrum (oxidized form) exhibited maxima at 280 nm and 400 nm; the A400/A280 ratio had a calculated value of 0.55. Chemical determination of its iron and sulfur atom content, the value of the extinction coefficient at 400 nm (epsilon 400 = 26.8 mM-1 cm-1) and EPR spectra indicated that ferredoxin II contained one [3Fe-4S] and one [4Fe-4S] cluster. Upon reduction with excess dithionite only the [3Fe-4S] cluster became reduced. The reduction of both clusters was achieved by using 5-deazaflavin as photocatalyst. Ferredoxin II was also purified from bacteria grown under nitrogen limiting (nif derepressing) conditions. In in vitro assays, ferredoxin II catalyzed electron transport between illuminated chloroplasts and nitrogenase.

Purification and properties of a halophilic catalase-peroxidase from Haloarcula marismortui

A heme protein, hCP, from the extreme halophile, Haloarcula marismortui, showing both peroxidatic and catalatic activity has been purified and characterized as a catalase-peroxidase. Catalatic activity is enhanced by molar concentrations of NaCl or (NH4)2SO4, while peroxidase activity decreases with increasing salt concentration. Optimal pH values are 6.0 for peroxidatic activity assayed in absence of NaCl and 7.5 for catalatic activity assayed in molar concentrations of NaCl. The two activities present saturation behaviour with increasing H2O2 concentration with apparent Km values of 0.5 and 2.5 mM for the peroxidatic and catalatic activities, respectively. A molecular mass of 81,292 +/- 9 Da was measured for the polypeptide by mass spectroscopy. One heme group (protoporphyrin IX with an iron atom in the ferric state) is associated with one molecule of hCP. Its amino-acid composition shows hCP to contain a high proportion of acidic residues. The EPR spectrum of the NO-compound of reduced (ferrous) hCP strongly suggests that the proximal ligand of the heme is the imidazole group of a histidine residue.

Molecular mechanism of pyruvate-ferredoxin oxidoreductases based on data obtained with the Clostridium pasteurianum enzyme

Pyruvate‐ferredoxin oxidoreductase oxidises pyruvate in many fermentative microorganisms. The enzyme from Clostridium pasteurianum is an air‐sensitive homodimer of 2×120000 daltons, for which pyruvate is the best substrate found among several α‐ketoacids. Each subunit contains eight iron atoms in two [4Fe‐4S] clusters. Two distinct EPR signals, possibly associated with two ligand environments, arise from one of these clusters. Binding of pyruvate does not generate a radical. The results reported suggest a scheme for the electron flow in pyruvate ferredoxin oxidoreductases according to which the detailed reaction mechanism depends on the number (even or odd) of [4Fe‐4S] clusters present in a given enzyme.

Intramolecular electron transfer in [4Fe-4S] proteins: estimates of the reorganization energy and electronic coupling in Chromatium vinosum ferredoxin

The semi-classical electron transfer theory has been very successful in describing reactions occurring in biological systems, but the relevant parameters in the case of iron-sulfur proteins remain unknown. The recent discovery that 2[4Fe-4S] proteins homologous to Chromatium vinosum ferredoxin contain clusters with different reduction potentials now gives the opportunity to study the dependence of the intramolecular electron transfer rate between these clusters as a function of the driving force. This work shows how decreasing the reduction potential difference between the clusters by site-directed mutagenesis of C. vinosum ferredoxin modifies the rate of electron hopping between the two redox sites of the protein by measuring the line broadening of selected 1H NMR signals. Beside the shifts of the reduction potentials, no signs of large structural changes or of significant alterations of the intrinsic kinetic parameters among the different variants of C. vinosum ferredoxin have been found. A reorganization energy of less than 0.5 eV was deduced from the dependence of the electron transfer rates with the reduction potential difference. This small value is associated with a weak electronic coupling between the two closely spaced clusters. This set of parameters, determined for the first time in an iron-sulfur protein, may help to explain how efficient vectorial electron transfer occurs with a small driving force in the many enzymatic systems containing a 2[4Fe-4S] domain.

Electron transfer properties of iron–sulfur proteins

The details of most electron transfer reactions involving iron-sulfur proteins have remained undisclosed because of the lack of experimental methods suitable to measure precisely the relevant rates. Nuclear magnetic resonance (NMR) provides a powerful means to overcome these problems, at least with selected proteins. A combination of NMR studies and site-directed mutagenesis experiments has been instrumental in defining both the site of interaction and the main trends of the intracomplex electron transfer in the case of rubredoxin electron self-exchange. Analysis of the NMR data obtained for mixtures of different redox levels of several 2[4Fe-4S] ferredoxins provided both first-order, for intramolecular, and second-order, for intermolecular, rate constants. Their dependence as a function of structural changes gave insight into the mechanism of electron transfer in this type of protein. Contrary to some expectations, the high-spin [4Fe-4Se]+ clusters assembled in isopotential ferredoxins do not change the intramolecular electron transfer rate as compared to low-spin [4Fe-4S]+ homologs. In combination with activity measurements, the kinetic data have been used to model the electron transfer competent complexes between Clostridium pasteurianum ferredoxin and the main enzymes acting as redox partners in vivo.