Patrick Bertrand

A theoretical interpretation of the variations of some physical parameters within the [2Fe-2S] ferredoxin group

A model is proposed to explain the variation of some physical parameters within the reduced [2Fe-2S] ferredoxin group. According to this model, the main effects result from a variable mixing of some d orbitals of the Fe2+ ion owing to rhombic distortion of the active site having the same geometrical character, but different in intensity, for each protein. Some peculiar experimental results such as the axial electron paramagnetic resonance spectra of adrenal ferredoxin and Pseudomonas putida ferredoxin and the electric field gradient tensor of P. putida ferredoxin are explained without assuming properties drastically different from those of the other ferredoxins, as had been suggested in the literature.

A ligand-field model to describe a new class of 2Fe-2S clusters in proteins and their synthetic analogues

Abstract Some metalloproteins which contain [2f-2S] clusters give EPR spectra characterized by g av ≈ 1.91, which is significantly lower than the value g av ≈ 1.96 usually obtained for the ferredoxins. A similar lowering of g av is observed for some [2F-2S] + thiolate synthetic compounds in glass-forming solvents, or in compounds where phenolates replace thiolates as terminal ligands. We show that a good correlation is observed for all the proteins and synthetic compounds characterized by g av ≈ 1.91, when the main components, g 1 , g 2 , g 3 , of the g tensor are plotted as a function of ( g 2 − g 3 ). This correlation supports the existence of a new class of [2F-2S] + clusters. A ligand-field analysis shows that the difference between the two kinds of EPR spectrum reflect essentially two different structures for the ferrous site. This conclucsion is in agreement with the spectroscopic studies performed on the Rieske protein from Thermus thermophilus .

Hydrogen‐Activating Enzymes: Activity Does Not Correlate with Oxygen Sensitivity

Hydrogenases (H2ases) catalyze a reaction that is essential in the energetics of many bacteria and has promising technological applications: the reversible conversion between dihydrogen and protons. These enzymes are studied in various contexts, including bioenergetics and inorganic catalysis, but the main motivation is that the knowledge we shall acquire by studying them will prove useful for designing the catalysts we need to produce hydrogen from water in a clean process and to oxidize it in affordable fuel cells. However, the fact that H2ases are inhibited by O2 hinders technological developments and much effort has been devoted to understanding the molecular basis of O2 sensitivity. [1, 2] Herein, we show that a very active H2ase, which was believed to be highly oxygen sensitive, actually reacts very slowly with O2 as a result of simultaneous reactions which we were able to resolve and quantify. Hydrogen-activating enzymes are classified as NiFe, Fe, and FeFe H2ases according to the metal content of their active site. Clostridial-type FeFe H2ases house four iron–sulfur (FeS) clusters in addition to the active-site “H-cluster”. Clostridium acetobutylicum (Ca) FeFe H2ase oxidizes H2 faster than the thoroughly characterized enzyme from Desulfovibrio desulfuricans (Dd), which has a similar structure except that the electron-transfer (ET) chain consists of two FeS clusters rather than four (Figure 1). We have used the procedure described in reference [3] to express homologously and purify by affinity chromatography to apparent homogeneity (as determined by sodium dodecylsulfate PAGE and elemental analysis) the strep-tagged FeFe H2ase (HydA1) from Ca (see the Supporting Information). This enzyme spontaneously adsorbs onto graphite in a configuration that allows direct ET to and from the electrode. The steady-state voltammograms in Figure 2 show that Ca

Application of EPR Spectroscopy to the Structural and Functional Study of Iron-Sulfur Proteins

Publisher Summary This chapter reviews the various applications of electron paramagnetic resonance (EPR) spectroscopy developed in the field of iron–sulfur research. As the electronic structure of the centers has often been explicitly dealt with in these studies, the main characteristics of the EPR spectrum and the relaxation properties of the diverse types of centers are presented in relation to their electronic structure. Some of the issues raised by the identification of iron–sulfur centers by EPR are discussed in the chapter. Various approaches, involving EPR spectroscopy, that have been used to obtain structural information about systems, containing iron-sulfur centers, are reviewed. These include elucidating their coordination scheme by site-directed mutagenesis, establishing the relative arrangement of centers coupled by spin-spin interactions, and determining their magnetic axes in oriented systems. Most of these structural investigations naturally have functional implications.

A needle in a haystack: The active site of the membrane-bound complex cytochrome c nitrite reductase

Cytochrome c nitrite reductase is a multicenter enzyme that uses a five‐coordinated heme to perform the six‐electron reduction of nitrite to ammonium. In the sulfate reducing bacterium Desulfovibrio desulfuricans ATCC 27774, the enzyme is purified as a NrfA2NrfH complex that houses 14 hemes. The number of closely‐spaced hemes in this enzyme and the magnetic interactions between them make it very difficult to study the active site by using traditional spectroscopic approaches such as EPR or UV–Vis. Here, we use both catalytic and non‐catalytic protein film voltammetry to simply and unambiguously determine the reduction potential of the catalytic heme over a wide range of pH and we demonstrate that proton transfer is coupled to electron transfer at the active site.

EPR potentiometric titration of c3-type cytochromes

The EPR potentiometric titration for multihaemic cytochromes requires the measurement of the intensity of the absorption EPR spectrum at different potential. We used this method to determine the midpoint potentials of two different cytochromes c3 isolated from Desulfovibrio desulfuricans Norway strain. The four haems of cytochrome c3 (Mr 13 000) have four different potentials (− 150, −270, −325 −355 mV), the highest value being well separated from the others. A similar conclusion was obtained from electrochemical measurements, although the values were slightly different (Bruschi, M., Loutfi, M.; Bianco, P. and Haladjian, J. (1984) Biochem. Biophys. Res. Commun. 120, 384–389). We discuss the origin of the discrepancy with the results from EPR titration which have been previously reported (Cammack, R., Fauque, G., Moura, J.J.G. and Le Gall, J. (1984) Biochim. Biophys. Acta 784, 68–74). For the dimeric cytochrome c3 (Mr 26 000), the midpoint potentials are also all different, with values ranging from −180 to − 390 mV. The relative contribution of the haems to several features of the derivative spectra is estimated for both proteins.

Single-crystal electron paramagnetic resonance study of cytochrome c3 from Desulfovibrio desulfuricans Norway strain : assignment of the heme midpoint redox potentials

A single crystal of cytochrome c3 from Desulfovibrio desulfuricans Norway is studied by electron paramagnetic resonance at low temperature. The orientation of the principal axis corresponding to the largest g value is determined for the 12 heme groups in the crystal unit cell. The comparison of these directions to the normals to the heme planes, determined from the crystallographic data at 2.5 A resolution, gives strong evidence for the following assignment of the midpoint redox potentials to the heme groups H1 to H4, defined in the three-dimensional structure: -150 mV is assigned to H3, -300 mV to H4, -330 mV to H1 and -355 mV to H2. This assignment is in agreement with a partial correspondence previously established from an independent study performed on cytochrome c3 in solution.

Temperature dependence of the electronic spin-lattice relaxation time in a 2-iron-2-sulfur protein

The ferredoxins are characterized by a strong temperature dependence of the electronic spin-lattice relaxation time T1. The measurement of this dependence above the liquid nitrogen temperature has been presented in earlier work [1] for the 2-iron-2-sulfur ferredoxin of the blue green alga Spirulina maxima. The different relaxation mechanisms which could be efficient in this range were briefly discussed. In the present paper, we extend the measurement of the temperature dependence of T1 to the low temperature range 1.25 to 30 K. From 1.25 K to 13 K, T1 is obtained by the saturating pulse method, whereas the continuous saturation method is used from 8 K to 30 K. The experimental conditions concerning these methods are discussed. The analysis of the temperature dependence curve over the whole range 1.25 K to 133 K shows clearly that different regions must be distinguished. For each region the possible relaxation processes and the corresponding vibrational modes are discussed.

EPR redox study of cytochrome c3 from Desulfovibrio vulgaris Miyazaki

We report the results of an EPR potentiometric titration of cytochrome c 3 from Desulfovibrio vulgaris Miyazaki: the EPR spectral features of the four hemes are identified. The four midpoint redox potentials, which are deduced from the integrated intensity variations as a function of the redox potential, are within the range −230 to −360 mV with two nearly equal intermediate values, in agreement with previous electrochemical measurements. A structural change of the environment of the heme with the most negative potential is observed during the first step of the reduction. The correspondence between the redox sites as characterized by the EPR potentiometric titration, and the hemes in the tridimensional structure, is discussed.

Electron spin–lattice relaxation of the (4Fe–4S) ferredoxin from B. stearothermophilus. Comparison with other iron proteins

The temperature dependence of the electron spin–lattice relaxation time T1 of the (4Fe–4S) ferredoxin from Bacillus stearothermophilus is studied in the range 1.2 to 40 K. This dependence is similar to that observed for the (2Fe–2S) ferredoxin from Spirulina maxima and can be interpreted with the same relaxation processes [J.P. Gayda, P. Bertrand, A. Deville, C. More, G. Roger, J.F. Gibson, and R. Cammack, Biochim. Biophys. Acta 581, 15 (1979)]. In particular, between 4 and 15 K, the data are well fitted by a second‐order Raman process involving three‐dimensional phonons, with a Debye temperature of about 60 K (45 cm−1). This would give an estimation of the highest frequency of the vibrations which can propagate through the three‐dimensional proteinic medium. In the highest temperature range (T≳30 K) the results are interpreted with an Orbach process involving an excited level of energy 120 cm−1. This process could be induced by the localized vibrations of the active site. Finally, these results are compa...