Dominique Job

Centre National de la Recherche Scientifique / French National Centre for Scientific Research, INSB, Emeritus

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Having been trained in Physics and Mathematics, I obtained a Master`s degree in Physics at the University of Dakar, Senegal. Back in France, I joined the laboratory of Plant Biochemistry (CNRS/ Marseilles University) to prepare a thesis during which I studied the physicochemical and enzymatic properties of various hemeproteins from plants, mainly by using rapid kinetic techniques (stopped-flow; T-Jump relaxation) and spectroscopic approaches at very low temperature. I was recruited at CNRS as a Research Associate in 1970. This work on hemeproteins was extended during two postdoctoral fellowships at the Department of Chemistry of the University of Edmonton, Canada, then at the University of Newcastle upon Tyne, UK.

In 1980, back in Marseilles, I started studying the mechanisms of transcription in plants. The major findings were the elucidation of the mechanisms contributing to the processivity of transcription RNA chain elongation, the factors influencing the balance between abortive and productive initiation, the mechanism of action of α-amanitin (a specific inhibitor of RNA pol II) and the influence of the sequence and conformation of the DNA template on the velocity and fidelity of transcription.
In 1992, I moved to Lyon to integrate the UMR041 CNRS/Rhône-Poulenc, a public-private joint laboratory, now the UMR5240 CNRS/Bayer CropScience, where I started working on seed physiology along with some research on the characterization of the enzymatic pathways that in plants are responsible for essential amino acid (sulfur amino acids, branch chain amino acids) and vitamin (biotin) metabolisms. I became successively deputy Head, then Head, then again deputy Head of this laboratory over the period 1993-2010. In particular, in 1999, I started a systematic study of the Arabidopsis (the model plant in genetics and genomics) seed proteome.

Beyond research in the laboratory, I have been, since 2000, involved in the promotion of plant genomics in France (Genoplante) and Europe (Plant KBBE, Plant 2030: Plant Biotechnology for the Future).

I am member of the Editorial Board of Molecular & Cellular Proteomics, Seed Science Research, Scientific Reports, and Associate Editor of Frontiers in Plant Science and of Proteomes. I am Emeritus CNRS Research Director, honorary consulting Professor at AgroParisTech and Member of the French Academy of Agriculture.

My current research work mainly deals with the study of seed physiology of Amborella trichopoda, the sister of all flowering plants.

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PEROXIDASES by Dominique Job

Substituent Effect on the Oxidation of Phenols and Aromatic Amines by Horseradish Peroxidase Compound I

vol 66, pp 607-614, 1976

A stopped-flow kinetic study shows that the reduction rate of horseradish peroxidase compound I b... more A stopped-flow kinetic study shows that the reduction rate of horseradish peroxidase compound I by phenols and aromatic amines is greatly dependent upon the substituent effect on the benzene ring. Moreover it has been possible to relate the reduction rate constants with the ionization constants of monosubstituted substrates by a linear free-energy relationship (Hammett equation). The correlation of log (rate constants) with (r values (Hammett equation) and the absence of correlation with CT' values (Okamoto-Brown equation) can be explained by a mechanism of aromatic substrate oxidations, in which the substrate gives an electron to the enzyme compound I and simultaneously loses a proton. The analogy which has been made with oxidation potentials of phenols or anilines strengthens the view that the reaction is only dependent on the relative ease of oxidation of the substrate. The rate constant obtained forp-aminophenol indicates that a value of 2.3 x 10' M-' s- ' probably approaches the diffusion-controlled limit for a bimolecular reaction involving compound I and an aromatic substrate.

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Horseradish peroxidase. XXVIII. Formation and reactivity of the alkaline form. Evidence for an enzyme–substrate complex in compound 1 formation

The rate of formation of 1 from horseradish peroxidase and hydrogen peroxide is investigated as a... more The rate of formation of 1 from horseradish peroxidase and hydrogen peroxide is investigated as a function of pH between pH 9.5 and 11.5. For pH values smaller than 10, the rate depends linearly on hydrogen peroxide concentration, whereas between pH 10 and 11.5, the rate displays saturation kinetics. Evidence is presented for the formation of an intermediate complex before compound 1 formation. Two parameters were measured as a function of pH: k,,,,, the apparent rate constant for conipound 1 formation, and k ,, the dissociation rate constant of the intermediate conlplex to 1. A third parameter, K;,,,,, similar to a dissociationconstant for the reaction was deduced from the two rate constants. The apparent rate constant k:,,,, is pH independent for pH values <9.5 and is directly affected by ionization of only onc group on the native enzyme with a pK of I I. The pH dependences of /id and K,,,, require a minimum of two ionizations. One corresponds to a group on the native enzyme which ionizes at a pH lower than that of the present study, the second corresponds to the ionizable group with pK of 11. For cyanide binding to horseradish peroxidase over the same pH interval, saturation kinetics were not observed, yet the pH profile for k,,, exactly parallels that for formation of 1 (when corrected for the effect of the ionization of HCN) and shows an inflection at the same pH value.

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Evolution of enzyme activity: Is diffusion control important? Activation parameters in the reactions of ferric heme species with hydrogen peroxide

The Journal of Physical Chemistry, 1993

There is a finite energy of activation for a diffusion-controlled reaction. It can be calculated ... more There is a finite energy of activation for a diffusion-controlled reaction. It can be calculated by comparing a combination of the Stokes-Einstein relation between the diffusion coefficient of a solute molecule and the viscosity of a solvent and the Smoluchowski equation for a diffusion-controlled rate constant to the rate equation of simple transition state theory. The entropy of activation for a diffusion-controlled reaction can also be obtained. Comparison is made of the enthalpy and entropy of activation for a diffusion-controlled reaction to the enthalpies and entropies of reaction of a series of heme-containing proteins and a simple heme with hydrogen peroxide. For nonenzymatic species the enthalpy of activation is greater than that for the ionization of hydrogen peroxide, indicating that they react with the conjugate base of hydrogen peroxide. For the peroxidases and catalases, both the enthalpy and entropy of activation are less than that for both hydrogen peroxide ionization and a diffusion-controlled reaction. The latter result indicates reaction with unionized hydrogen peroxide, which gives the enzymes a huge advantage over the nonenzymatic species at physiological pH. It also indicates that the peroxidases and catalases do not operate by a diffusion-controlled mechanism. Our conclusion is that the hypothesis of the diffusion-controlled limit being the ultimate criterion of a perfectly evolved enzyme is too restrictive. Rather, enzymes evolve in such a manner that they ultilize the binding energy of the substrate to lower the enthalpy of activation below that for a diffusion-controlled reaction, in compensation for the necessary low entropy of activation required to form a transition state which is much more highly ordered than that of the separated reactants.

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Comment on “Thermodynamic Trajectory of Enzyme Evolution”

The Journal of Physical Chemistry B, 1997

Chemistry have addressed the question of whether the ultimate target of enzyme evolution is a dif... more Chemistry have addressed the question of whether the ultimate target of enzyme evolution is a diffusion-controlled reaction. In the earlier paper, 1 the temperature dependencies of rates of reactions of a series of heme proteins with hydrogen peroxide were analyzed in terms of ∆H q and ∆S q . Globins had ∆H q and ∆S q values much greater than that for an aqueous diffusioncontrolled reaction, and peroxidases and catalases had smaller values. The globins have similar structures to the peroxidases and catalases, and if they are not evolutionary precursors, there would at least appear to be a common evolutionary ancestor. 2 In the sequel paper, 3 the same kinetic data were reanalyzed on the basis of a series of assumptions that led the authors to conclude that a corner could be turned and the diffusioncontrolled target could be reached, despite being initially bypassed.

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Compound I Formation with Turnip Peroxidases and Peroxybenzoic Acids

European Journal of Biochemistry, 1978

The kinetics of formation of the compounds I of the turnip peroxidase isoenzymes 1 and 7 with per... more The kinetics of formation of the compounds I of the turnip peroxidase isoenzymes 1 and 7 with peroxybenzoic acid and a series of substituted peroxybenzoic acids (p-OCH3, p-CH3, p-C1, mC1, rn-NO2 andp-N02) were studied at 25 "C. The pH profiles of the observed second-order rate constants, after correction for the alkaline transitions of the enzymes, indicate that isoenzyme 1 reacts exclusively with unionized peroxy acids whereas with isoenzyme 7, although the major contribution involves reaction with unionized peroxy acid, an additional reaction with peroxy anion is observed. In contrast to the behaviour of horse-radish peroxidase, (where the rate constants for unhindered unionized peroxybenzoic acids exceed that with H202 and the reactions are probably diffusion controlled) the rate constants, kHAf,o r isoenzyme 1 reaction with unionized peroxy acids are all lower than that with H202 and the activation energies indicate chemical control. For isoenzyme 7 the value of HA with the most weakly acidic peroxybenzoic acids exceeds the rate constant with H202. For both isoenzyme 1 and isoenzyme 7 the values of kHA are very sensitive to peroxy acid acidity and in both cases log kHA shows good correlation with PKHA. The results are compared with each other and with previous data for both turnip and horse radish peroxidases. It is suggested that the multifactorial influences upon the kinetics of compound I formation include (a) diffusion, (b) substrate hydrophobicity, the hydrophobic binding affinity of the active sites of the enzymes and perhaps hydrogen bonding interactions, (c) a substrate-charge-type discrimination elicited by the protein at the entrance to the active site, (d) substrate substituent inductive effects which suggest the importance of the generation of highly nucleophilic peroxy anions from neutral hydroperoxides within the active site. These influences are of very variable relative importance in enzymes from different sources and in different isoenzymes from the same source.

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Kinetics of formation of the primary compound (compound I) from hydrogen peroxide and turnip peroxidases

Canadian Journal of Biochemistry, 1978

the apparent rate constant for conipound 1 formation, and k,, the dissociation rate constant of t... more the apparent rate constant for conipound 1 formation, and k,, the dissociation rate constant of the intermediate conlplex to 1. A third parameter, K;,,,,, similar to a dissociatio~i constant for the reaction was deduced from the two rate constants. The apparent rate constant k:,,,, is p H independent for p H values < 9 . 5 and is directly affected by ionization of only o n c group on the native enzyme with a pK of I I. The p H dependences of /id and K,,,, require a minimum of two ionizations. One corresponds to a group on the native enzyme which ionizes at a p H lower than that of the present study, the second corresponds t o the ionizable group with pK of 11. F o r cyanide binding to horseradish peroxidase over the same p H interval, saturation kinetics were not observed, yet the p H profile for k,,, exactly parallels that f o r formation of 1 (when corrected for the effect of the ionization of HCN) a n d shows an inflection at the same p H value.

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Substituent Effect on the Oxidation of Phenols and Aromatic Amines by Horseradish Peroxidase Compound I

European Journal of Biochemistry, 1976

A stopped-flow kinetic study shows that the reduction rate of horseradish peroxidase compound I b... more A stopped-flow kinetic study shows that the reduction rate of horseradish peroxidase compound I by phenols and aromatic amines is greatly dependent upon the substituent effect on the benzene ring. Moreover it has been possible to relate the reduction rate constants with the ionization constants of monosubstituted substrates by a linear free-energy relationship (Hammett equation). The correlation of log (rate constants) with (r values (Hammett equation) and the absence of correlation with C T ' values (Okamoto-Brown equation) can be explained by a mechanism of aromatic substrate oxidations, in which the substrate gives an electron to the enzyme compound I and simultaneously loses a proton. The analogy which has been made with oxidation potentials of phenols or anilines strengthens the view that the reaction is only dependent on the relative ease of oxidation of the substrate. The rate constant obtained forp-aminophenol indicates that a value of 2.3 x 10' M-' s-' probably approaches the diffusion-controlled limit for a bimolecular reaction involving compound I and an aromatic substrate.

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Circular dichroism of turnip peroxidases

Canadian Journal of Biochemistry, 1977

Circular dichroic (CD) spectra of two turnip isoperoxidases, P1 and P7, and of their derivatives ... more Circular dichroic (CD) spectra of two turnip isoperoxidases, P1 and P7, and of their derivatives were measured over the wavelength range of 200 to 650 nm. For the two isoenzymes, it was observed that although the visible and Soret bands are located at similar wavelengths, their ellipticities are different. These results suggest that the active sites are similar but that differences do exist. The results are compared with those reported for Japanese radish peroxidase a and horseradish peroxidase. It appears that a common property of plant peroxidases is the presence of negative CD Soret bands for the reduced forms and their inversion upon cyanide binding. The CD spectra in the far UV region indicate an appreciable helical content for both native enzymes and their various derivatives. The calculated contents of unordered structure are very high (greater than 50% for either P1 or P7), in agreement with other studies on glycoproteins.

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Resonance Raman study of plant tissue peroxidases Common characteristics in iron coordination environments

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1983

Resonance Raman spectra of isozymes 2, 3, 9, 15, and 16 of Japanese radish peroxidase and isozyme... more Resonance Raman spectra of isozymes 2, 3, 9, 15, and 16 of Japanese radish peroxidase and isozymes 1, 3,
and 7 of turnip peroxidase were measured and compared with those of horseradish peroxidase. All the resting
isozymes gave the J'10 band around 1629-1631 cm-1, indicating the five-coordinate high-spin structure. The
reduced form of all these isozymes provided the iron-histidine stretching Raman line at distinctly higher
frequencies in comparison with those of hemoglobin and exhibited a clear pH-dependent frequency shift at
neutral pH in accord with the results for horseradish peroxidase. Therefore, we conclude that strong
hydrogen bonding of the proximal histidine and a small structural change of the proximal histidine at neutral
pH without breakage of the hydrogen bond are the common characteristics of plant tissue peroxidases (EC
1.11.1.7) which contrast with those of oxygen-carrier hemoproteins.

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