Claude More

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 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 (0.8–100u2009cm−1) for the parameter J0 of the isotropic interaction J0SA.SB, but neither the magnitude of J0 nor its sign could be further specifie...

g̃‐tensor calculation for FeIII triads: Application to the 3Fe clusters in iron–sulfur proteins

The g tensor of a triad of high spin FeIII ions coupled by antiferromagnetic exchange interactions, is calculated in the case where the fine structure terms are not negligible compared to the exchange parameters. Using some simplifying assumptions concerning the relative orientations of the magnetic axes, we obtain the expressions of the g components in closed form. A simple ‘‘g strain’’ model based on these expressions is used to simulate the EPR spectra given by the 3Fe clusters of four different iron sulfur proteins. In each case, a good simulation is only observed if the three exchange parameters are nearly equal, and of the order of −20 cm−1. These conclusions are in good agreement with our earlier estimations which were obtained by an independent method [J. P. Gayda, P. Bertrand, F‐X. Theodule, and J. J. G. Moura, J. Chem. Phys. 77, 3387 (1982)].

Interconversions between the 3Fe and 4Fe forms of the iron-sulfur clusters in the ferredoxin from Thermodesulfobacterium commune: EPR characterization and potentiometric titration

Abstract The amount of 3Fe clusters in Thermodesulfobacterium commune ferredoxin is strongly dependent upon the presence of oxygen during the purification. An average of one 3Fe cluster per monomer can be found when the purification is not strictly anaerobic. These clusters are converted into |4Fe-4S| clusters by adding dithionite at usual pH and without adjunction of Fe 2+ . The EPR potentiometric titration reveals the existence of several types of 3Fe clusters with negative midpoint potentials differing by more than 100 mV. When the |4Fe-4S| clusters are partially reduced the EPR signal is composed of two different rhombic components. The component with g z = 2.04 could be related to a site implicated in the interconversion processes. In the fully reduced state, the spectrum presents the typical features of two interacting |4Fe-4S| clusters as those observed in two |4Fe-4S| bacterial ferredoxins. From the redox titration curves the midpoint potentials of these clusters are estimated at −395 and −435 mV.

Relationship between structural and magnetic properties of the 3Fe clusters in iron-sulfur proteins

Abstract We show that the main properties of the EPR spectrum given by the 3Fe clusters of iron-sulfur proteins are well described if the fine structure terms are considered in the calculation of the ḡ-tensor. This model accounts for the particular asymmetry of the spectrum at low temperature and for the g values well below 2 which are observed in the high-field region. A good agreement with the experimental data is only obtained if the three exchange parameters are nearly equal, with values of about −20 cm −1 . This supports our earlier estimations which were derived by an independent method. The implications of these results for the models which have been proposed to describe the 3Fe cluster structure are discussed.

Simulation of the EPR spectra of metalloproteins based on a physical description of the “g-strain” effect

Abstract The EPR spectra given by metalloproteins in frozen solution are characterized by a large broadening due to a distribution of conformations, the so-called “g-strain” effect. For some classes of metalloproteins, models are available to describe the magnetic properties of the active sites, and the g values can be expressed in terms of physical parameters. The distribution of conformations can then be accounted for by allowing some of these parameters to be random variables, and the EPR signal is obtained by a superposition of the powder spectra corresponding to a given set of values of random variables. We have applied this procedure to reduced 2Feue5f8-2S ferredoxins, and shown that the theoretical model which accounts for the variations of the g values within the 2Feue5f82S class can also be used to describe the conformational states of a given protein.

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

Abstractu2002The 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.