Richard H. Sands

The iron electron-nuclear double resonance (ENDOR) of two-iron ferredoxins from spinach, parsley, pig adrenal cortex and Pseudomonas putida ☆

Abstract The iron electron-nuclear double resonance (ENDOR) spectra of reduced iron-sulfur proteins (two-iron ferredoxins) from spinach, parsley, pig adrenal cortex and Pseudomonas putida unequivocally show two inequivalent iron atoms at the active sites of each of these proteins. The frequencies of the ENDOR lines establish the total electronic spin in the ground state to be S = 1 2 . The principal values of the hyperfine tensor have been determined for each of the iron atoms and these values are consistent with and lend considerable support to the model of a high-spin Fe(III) atom and a high-spin Fe(II) atom antiferromagnetically coupled to form an S = 1 2 system. The measured principal axis components of the effective hyperfine tensors for S = 1 2 are as follows (1 and 2 refer to the inequivalent iron sites): Site 1 ( Fe ( III )) Site 2 ( Fe ( II )) A x A y ′ A z ′ A x A y A z Spinach 51 ± 1 50 − 7 +2 42 ± 1.5 ? ? 35.5 ± 2 MHz Parsley 51 ± 1 50 − 7 +2 42 ± 2 ? ? 34.5 ± 2.5 MHz A ⊥ A ⊥ ′ A z A ⊥ A ⊥ ′ A z Adrenodoxin 50 ± 1 56 − 3 +1 43 +2 − 1 17 ± 4 24 ∓ 4 35 ± 1.5 MHz Putidaredoxin 50 ± 1.5 56 − 3 +1 43 +2 − 1 17 ± 4 24 ∓ 4 35 ± 1.5 MHz These data are consistent with Site 1 being ferric and Site 2, ferrous iron. The primes indicate that the A -tensor principal axes for Site 1 (Fe(III)) are apparently rotated about the x -axis with respect to the g -tensor axes by an angle θ (20° ⩽ θ ⩽ 40°). The orientations of the A -tensors for Site 2 (Fe(II)) have not been determined and hence the values presented are the observed values of the A -tensors along the x , y , and z -axes of the g -tensor for this complex. A brief introduction to the theory of ENDOR is given.

The two-iron ferredoxins in spinach, parsley, pig adrenal cortex, Azotobacter vinelandii, and Clostridium pasteurianum: Studies by magnetic field Mössbauer spectroscopy☆

Abstract The two-iron ferredoxins from spinach, parsley, Azotobacter vinelandii, Clostridium pasteurianum and the pig adrenal cortex were investigated by Mossbauer spectroscopy at temperatures from 4 to 256°K and in magnetic fields up to 46 kGauss. Computational programs were devised to allow comparison of the experimental data with computer-simulated spectra in order to facilitate identification of the experimental spectral detail with specific Mossbauer spectroscopic parameters (quadrupole splittings, isomer shifts and nuclear hyperfine and nuclear Zeeman interactions). The results of the analysis permit the following properties of the active center to be established directly as the result of these experiments: 1. 1. In the oxidized forms of the proteins, each iron is in the high spin ( S = 5 2 ) ferric state, spin-coupled to produce a resultant molecular diamagnetism for the protein at temperatures below 100°K. 2. 2. In the reduced state of the protein, the active center contains a single ferric site, retaining many properties of the ferric iron in the oxidized protein, but spincoupled to a high spin ( S = 2) ferrous site, producing a molecular paramagnetism due to a net electron spin of one half at low temperatures ( S = 1 2 ). 3. 3. In spinach and parsley ferredoxins, the ligand symmetry around the ferrous site in the reduced form of the proteins is tetrahedral with measurable axial and rhombic distortions. 4. 4. The iron sites in both the oxidized and reduced forms of all the proteins studied are similar, with the possible exception that the ligand symmetry at the ferrous site in the reduced form of the two-iron ferredoxins from C. pasteurianum, A. vinelandii (Azotobacter I and II), and pig adrenal cortex has not been characterized as being square planar or tetrahedral, although octahedral symmetry has been excluded.

On the structure of the iron-sulfur complex in the two-iron ferredoxins

Abstract Recent spectroscopic and magnetic susceptibility studies of the iron center in the two-iron ferredoxins provide criteria which any model for the iron-sulfur complex in these proteins must satisfy. These criteria are most stringent for parsley and spinach ferredoxin: the reduced proteins contain a high-spin ferric atom antiferromagnetically exchange-coupled (presumably via sulfide bridging ligands) to a high-spin ferrous atom. In the oxidized proteins the iron atoms are antiferromagnetically spin-coupled, high-spin ferric atoms. Arguments are given to substantiate the claim that the ferrous atom in the reduced protein is ligated by four sulfur atoms in a distorted tetrahedral configuration: two are the bridging sulfides, two are cysteinyl sulfurs. A treatment of proton contact shifts based upon the above model is pertinent to proton magnetic resonance data already available and provides a means to identify directly the ligands at both iron atoms via further PMR experiments.

The magnetic susceptibility of spinach ferredoxin from 77–250°K: A measurement of the antiferromagnetic coupling between the two iron atoms☆

Abstract The magnetic susceptibility of oxidized and reduced spinach ferredoxin has been measured over the temperature range 77–250°K. Anomalous behavior is observed in both oxidation states and the data can be interpreted by assuming an exchange interaction between the metal ions. The exchange constant is estimated to be 183 cm −1 in oxidized ferredoxin and ≤ 100 cm −1 in reduced ferredoxin.

A Statistical Theory for Powder EPR in Distributed Systems

p, whose principal elements are random variables. The p and g tensors are not necessarily colinear. The observed EPR linewidth results from a distribution in the effective g value as a function of (a) the joint distribution function of the elements of the p tensor and (b) the spatial relationship between the two principal axis systems involved. The theory is reformulated in terms of matrices that facilitate a direct comparison with earlier work. Two previous theories of g strain represent different subsets of the general theory, namely, the case of zero rotation between axis systems and the case with nonzero rotation and full correlation between elements of the p

On the nature of the iron sulfur cluster in a deuterated algal ferredoxin

A protonated and a completely deuterated two-iron algal ferredoxin from Synechococcus lividus have been studied by optical, electron paramagnetic resonance, electron-nuclear double resonance, proton magnetic resonance and Mossbauer spectroscopies; temperature dependent magnetic susceptibility measurements are reported as well. These studies have confirmed the electron localized model of the active center in the two-iron ferredoxins, as previously deduced from studies of spinach ferredoxin, have yielded much more precise spectroscopic parameters for this center, and have thus greatly increased the confidence in this model.

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.

An investigation of Chromatium vinosum high-potential irondashsulfur protein by EPR and Mossbauer spectroscopy; evidence for a freezing-induced dimerization in NaCl solutions

The high-potential iron-sulfur protein (HiPIP) from Chromatium vinosum contains a cubane prosthetic group that shuttles between the [4Fe-4S]3+,2+ states. We find that the EPR spectra from this protein can be explained as a sum of two components, a major one with g = 2.02; 2.04; 2.12, and a minor one with g = 2.04; 2.07; approximately 2.13. In the presence of 0.1-2.0 M NaCl, freezing induces polymerization of the protein (presumably dimers), which is detected as intercluster spin-spin interaction in the EPR. The observed spin-spin interactions are interpreted as being due to two very similar dimeric structures in an approx. 1:2 ratio. Computer simulation of the X- and Q-band EPR spectra shows that the z-components of the g-tensors in each dimer pair must be co-linear, with center-to-center distances between the clusters of approximately 13 A and approximately 16 A. Inspection of possible dimeric structures of C. vinosum HiPIP by standard molecular graphics procedures revealed that the Fe/S cluster is exposed toward a flattened surface and is accessible to solvent. Moreover, the Fe/S clusters in two HiPIP molecules can easily achieve a center-to-center distance of approximately 14 A when approaching along a common 3-fold axis that extends through the S4 sulfur atom of the cubane; the z-component of the EPR g-tensor is co-linear with this symmetry axis.

Electron–nuclear double resonance on copper (II) tetraimidazole

We have investigated the electron–nuclear double resonance (ENDOR) from frozen aqueous solutions of 65Cu++(imidazole)4, 65Cu++ (imidazole–15N)4, and 65Cu++(imidazole–Dn)4, where n = 1, 2, 3, and 4 for selectively deuterated imidazole. We have observed ENDOR associated with the imidazole protons and the two imidazole nitrogens. The selective deuteration has allowed us to attempt identification of the weakly coupled protons responsible for the ENDOR spectrum, and a comparison of the overall line shape of that spectrum taken at two extreme points of the EPR spectrum suggests that some of the imidazole planes are tilted with respect to the plane of the complex. The ENDOR arising from the nitrogen nearest the copper is primarily isotropic with A(g⊥) = 41.6±1.5 MHz and A(g∥) = 39.8±1.5 MHz. The resonance shows little structure and seems consistent with a picture that requires some inequivalence among the various imidazoles. The remote nitrogen ENDOR reveals both hyperfine and quadrupole effects with approximate...

An Analysis of g Strain in the EPR of Two (2Fe-2S) Ferredoxins. Evidence for a Protein Rigidity Model

Abstract Replacing current notions of a paramagnetic center in a metalloprotein as a single entity in vivo with the more realistic concept of an ensemble of spin systems, each uniquely disturbed by its own surrounding protein, leads to a rigorous description of the spectroscopic factor, g, as a random variable whose statistical properties contain information on the rigidity of the protein. Generation of a consistent set of accurate simulations of very low-noise, multifrvquency (3, 9, 15 GHz) EPR data from selected S = 1 2 proteins has now been achieved. This consistency lends support to the physical and biological inferences drawn from such simulations. The spectral contribution of magnetic hyperfine line-broadening is minimized by studying the 56Fe reconstituted [2Fe2S] cluster in fully deuterated ferredoxin from Synechococcus lividus and the 2H2O exchanged [2Fe2S] ferredoxin from Pseudomonas putida. High-resolution Mossbauer data on oxidized and reduced 57Fe reconstituted S. lividus ferredoxin are also presented. The oxidized spectrum shows that the inequivalence of the two ferric ions in a [2Fe2S] cluster can be resolved as two Mossbauer lines. The complete absence of this splitting in the ferric fines of the reduced spectrum is definitive proof that the reducing electron always resides at the same 56Fe atom in frozen aqueous solutions. To explain the distributed nature of the paramagnetic site in the ferredoxins, three models are considered: (1) a multiplicity of EPR states; (2) external perturbations to the molecular Hamiltonian; (3) a distribution in the crystal field Hamiltonian parameters. The first model is discarded, the second is possible but difficult to verify, and the third model is shown to fit the data well. The latter comparison requires a correction to literature expressions for the g and A tensors in [2Fe2S] clusters. Statistical analysis strongly suggests that the EPR of metalloproteins in its details is a reflection of protein structure that distributes its spatial coordinates, accommodating different levels of rigidity, the more flexible parts being located at the outside.