Roland Aasa

EPR signal intensity and powder shapes: A reexamination

Integrated intensities of EPR lines and the simulation of powder shapes in the presence of large anisotropy are discussed for field-sweep spectra. It is pointed out that the shape function, normalized by integration over the magnetic field, must be multiplied by a factor which in the S = 12 case equals the inverse of the g-value. This factor seemingly has been omitted in previous calculations of intensities and shapes. As a consequence, the integrated intensity of an isotropic line is proportional to its g-value and not to g2. The total intensity of a powder spectrum is calculated, and examples of simulations of such spectra are given. A method for the determination of total intensities from the area under an “absorption” peak in a first derivative powder spectrum is also given.

Powder Line Shapes in the Electron Paramagnetic Resonance Spectra of High‐Spin Ferric Complexes

The positions of powder lines in the electron paramagnetic spectra of high‐spin ferric systems (d5, S = 52) have been calculated by solving the spin Hamiltonian H = gβB·S + 13D[3Sz2 − S(S + 1)] + E(Sx2 − Sy2) for a broad range of parameters. Powder lines are obtained for every transition when the magnetic field points along the principal axes of the fine structure tensor. However, it was found that for most transitions extra powder lines are often found when the field lies in any of the principal planes but not along the axes. Particular attention is directed to the transition responsible for the g′ ≈ 4.2 absorption in nearly rhombic (E / D∼13) ferric complexes. The calculations show that, depending on the value of the ratio between the microwave quantum and the parameter D, this transition may consist of 3–6 powder lines near g′ = 4.2. The g′ values for all these powder lines were also obtained from a third‐order perturbation calculation which assumes nearly rhombic symmetry and D > gβB. The 9.2‐ and 34‐...

The Origin of the Multiline and g=4.1 Electron Paramagnetic Resonance Signals from the Oxygen-Evolving System of Photosystem II

Continuous illumination at 200 K of photosystem (PS) II-enriched membranes generates two electron paramagnetic resonance (EPR) signals that both are connected with the S(2) state: a multiline signal at g 2 and a single line at g = 4.1. From measurements at three different X-band frequencies and at 34 GHz, the g tensor of the multiline species was found to be isotropic with g = 1.982. It has an excited spin multiplet at approximately 30 cm(-1), inferred from the temperature-dependence of the linewidth. The intensity ratio of the g = 4.1 signal to the multiline signal was found to be almost constant from 5 to 23 K. Based on these findings and on spin quantitation of the two signals in samples with and without 4% ethanol, it is concluded that they arise from the ground doublets of paramagnetic species in different PS II centers. It is suggested that the two signals originate from separate PS II electron donors that are in a redox equilibrium with each other in the S(2) state and that the g = 4.1 signal arises from monomeric Mn(IV).

Chlorite dismutase from Ideonella dechloratans

Chlorite dismutase has been purified from the chlorate-metabolizing bacterium Ideonella dechloratans. The purified enzyme is tetrameric, with a relative molecular mass of 25,000 for the subunit, and contains about 0.6 heme/subunit as isolated. Its catalytic properties are similar, but not identical, to those found for a similar enzyme purified earlier from the bacterium GR-1. The heme group in Ideonella chlorite dismutase is readily reduced by dithionite, in contrast to the GR-1 enzyme, and redox titration gave a value of –21 mV for the midpoint potential at pH 7. The heme group has been characterized by optical and EPR spectroscopy. It is high-spin ferric at neutral pH, with spectroscopic properties similar to those found for cytochrome c peroxidase. In the alkaline pH range, a low-spin compound is formed. A 22-residue N-terminal amino acid sequence has been determined and no homologue has been found in the protein sequence databases.

Properties and function of the two hemes in Pseudomonas cytochrome c peroxidase

The oxidation-reduction potentials of the two c-type hemes of Pseudomonas aeruginosa cytochrome c peroxidase (ferrocytochrome c:hydrogen-peroxide oxidoreductase EC 1.11.1.5) have been determined and found to be widely different, about +320 and -330 mV, respectively. The EPR spectrum at temperatures below 77 K reveals only low-spin signals (gz 3.24 and 2.93), whereas optical spectra at room temperature indicate the presence of one high-spin and one low-spin heme in the enzyme. Optical absorption spectra of both resting and half-reduced enzyme at 77 K lack features of a high-spin compound. It is concluded that the heme ligand arrangement changes on cooling from 298 to 77 K with a concomitant change in the spin state. The active form of the peroxidase is the half-reduced enzyme, in which one heme is in the ferrous and the other in the ferric state (low-spin below 77 K with gz 2.84). Reaction of the half-reduced enzyme with hydrogen peroxide forms Compound I with the hemes predominantly in the ferric (gz 3.15) and the ferryl states. Compound I has a half-life of several seconds and is converted into Compound II apparently having a ferric-ferric structure, characterized by an EPR peak at g 3.6 with unusual temperature and relaxation behavior. Rapid-freeze experiments showed that Compound II is formed in a one-electron reduction of Compound I. The rates of formation of both compounds are consistent with the notion that they are involved in the catalytic cycle.

EPR studies on compound I of horseradish peroxidase

Compound I of horseradish peroxidase (donor: hydrogen-peroxide oxidoreductase EC 1.11.1.7) was studied by EPR at low temperatures. An asymmetric signal was found, about 15 Gauss wide and with a g-value of 1.995, which could be detected only at temperatures below 20 K and which had an intensity corresponding to about 1% of the heme content. In a titration with H2O2, the signal intensity was proportional to the concentration of Compound I, reaching a maximum when equivalent amounts of H2O2 were added. This indicates that the signal is not due to an impurity, and it is suggested that a free radical is formed, relaxed by a near-by fast-relaxing iron.

Rack-induced bonding in blue copper proteins: spectroscopic properties and reduction potential of the azurin mutant Met-121 Leu

Site‐directed mutagenesis has been used to prepare azurin in which the methionine‐121 residue has been replaced by leucine. The oxidized mutant protein displays the strong blue color and characteristic EPR signal of a type 1 Cu(II) ion, showing that methionine is not an obligatory component of a blue copper site. The optical absorption maximum is shifted 5 nm towards longer wavelength and the extinction coefficient increased by about 10% compared to the wild‐type protein. In addition, there are small changes in the EPR parameters, in particular the copper hyperfine splitting. The reduction potential is increased by 70 mV. The results show that a small change in primary structure without any alteration in the three strong ligands can perturb the Cu(II) site and shift the reduction potential, in accord with the concept of rack‐induced bonding in blue copper proteins.

EPR Spectroscopy of soybean lipoxygenase-1: Description and quantification of the high-spin Fe(III) signals

The interaction of soybean lipoxygenase-1 with 13-Ls-hydroperoxy-9-cis,11-trans-octadecadienoic acid, the product of the enzymic dioxygenation of linoleic acid, results in the formation of either a yellow or a purple coloured enzyme form depending on the amount of product used. The composition of the high-spin Fe(III) signals in the EPR spectra of both enzyme forms has been studied and the amount of EPR-visible iron determined by integration and simulation. Sets of g values of the species building up the high-spin Fe(III) signal around g 6 are derived from both third-order perturbation calculation and exact numerical diagonalization of the spin Hamiltonian describing the system. The results of these calculations are generally applicable to systems having S = 5/2. The iron in the native, colourless enzyme is almost EPR-nondetectable. The yellow form of the enzyme shows a complex EPR signal around g 6 which consists of contributions of at least three species with different ligand symmetry. The signal corresponds to approx. 75% of the total iron content. The g 6 signal of the purple Fe(III) enzyme corresponds roughly to the same amount of iron but the ratio between the different species is not the same as in the yellow enzyme. This enzyme form also shows an additional g 4.3 signal with a large amplitude but a relatively low integrated intensity (approx. 10% of the total iron content). The results are consistent with the suggested mechanism of the catalytic function of iron in lipoxygenase which was based on qualitative EPR results (De Groot, J.J.M.C., Veldink, G.A., Vliegenthart, J.F.G., Boldingh, J., Wever, R. and van Gelder, B.F. (1975) Biochim. Biophys. Acta 377, 71--79).

The nature of the CuAcenter in cytochrome c oxidase

The merits of the suggestion that cuA in cytochrome oxidase is a mixed‐valence binuclear site is reviewed on the basis of recent analytical and spectroscopic studies. First an alternative mononuclear model is presented. Metal analyses indicate that homogeneous oxidase preparations with high activity contain 3Cu/2Fe. Multifrequency EPR measurements demonstrate a close similarity with a copper site in nitrous oxide reductase, and this is also supported by optical and MCD spectra. Strong evidence for a binuclear site is provided by a 7‐line hyperfine structure in the EPR spectra of both enzymes. A binuclear model consistent with amino acid sequence data can be formulated.

Multifrequency EPR investigations into the origin of the S2-state signal at g = 4 of the O2-evolving complex

The low-temperature S2-state EPR signal at g = 4 from the oxygen-evolving complex (OEC) of spinach Photosystem-II-enriched membranes is examined at three frequencies, 4 GHz (S-band), 9 GHz (X-band) and 16 GHz (P-band). While no hyperfine structure is observed at 4 GHz, the signal shows little narrowing and may mask underlying hyperfine structure. At 16 GHz, the signal shows g-anisotropy and a shift in g-components. The middle Kramers doublet of a near rhombic S = 5/2 system is found to be the only possible origin consistent with the frequency dependence of the signal. Computer simulations incorporating underlying hyperfine structure from an Mn monomer or dimer are employed to characterize the system. The low zero field splitting (ZFS) of D = 0.43 cm-1 and near rhombicity of E/D = 0.25 lead to the observed X-band g value of 4.1. Treatment with F- or NH3, which compete with Cl- for a binding site, increases the ZFS and rhombicity of the signal. These results indicate that the origin of the OEC signal at g = 4 is either an Mn(II) monomer or a coupled Mn multimer. The likelihood of a multimer is favored over that of a monomer.