Alan J. Bearden

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.

Quantitative EPR studies of the primary reaction of Photosystem I in chloroplasts

Abstract Quantitative electron paramagnetic resonance studies of the primary event associated with Photosystem I in chloroplasts have been carried out at 25 °K. After illumination of either whole chloroplasts or Photosystem I subchloroplast fragments (D-144) with 715-nm actinic light at 25 °K, equal spin concentrations of oxidized P700 and reduced bound iron-sulfur protein (bound ferredoxin) have been measured. Quantitative determination of the concentration of these two carriers by EPR spectroscopy after illumination at low temperature indicates that Photosystem I fragments are enriched in P700 and the bound iron-sulfur protein as compared with unfractionated chloroplasts. These results indicate that P700 and the bound iron-sulfur protein function as the donor-acceptor complex of chloroplast Photosystem I.

Imaging and vibrational analysis with laser-feedback interferometry.

We have constructed a scanning confocal laser-feedback microscope that determines surface profiles in the range of 5 nm to 3 μm with ~200-nm lateral discrimination. A direct comparison is made with scanning electron microscopy, and an image of a silicon resolution standard with 40-nm-high structures is shown. The device is also capable of characterizing surface motion with a sensitivity of <1 pm (Hz)−1/2 across a bandwidth of several megahertz. Vibrational analysis of a miniature piezoelectric microphone is demonstrated from 50 Hz to 50 kHz. An operational description of the device is presented in addition to a generalization of laser-feedback theory that includes gas-laser dynamics.

Primary photochemical reactions in chloroplast photosynthesis

Photosynthesis begins with the absorption of light energy and this absorbed energy is transferred to special sites, termed reaction centres. At these sites, the light energy is transformed into chemical products through an oxidation-reduction reaction that generates the primary reactants, an oxidized pigment molecule (P + ) and a reduced electron acceptor (A – ) (Clayton, 1972). The subsequent reactions of these species in the dark ultimately results in the formation of chemical products required for the fixation of CO 2 . In this essay we will discuss the nature of the primary reactants generated in the light reactions of chloroplast photosynthesis, stressing recent advances in the identification and characterization of such reactants.

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.

Light-induced changes of bound chloroplast plastocyanin as studied by EPR spectroscopy: The role of plastocyanin in noncyclic photosynthetic electron transport

Abstract A low-temperature electron paramagnetic resonance (EPR) signal in spinach chloroplasts has been identified as originating from oxidized bound plastocyanin. The EPR parameters of the bound plastocyanin, which are the same as those of soluble plastocyanin, are: g ⊥ = 2.05, g ∥ = 2.23, and A ∥ = 0.006 cm −1 . An estimation of the amount of bound plastocyanin based on the in situ EPR signal indicates a concentration of approximately 3 nmoles per mg chlorophyll, a value which agrees with the amount of plastocyanin determined by chemical assay after release from the chloroplast lamellae by sonication. Light-induced changes of plastocyanin in situ have been examined after illumination with monochromatic light at physiological temperatures. The bound plastocyanin undergoes a photoreduction in Photosystem II light which is sensitive to inhibitors of noncyclic electron transport. The presence of the NADP + acceptor system in Photosystem II light causes a shift in the steady-state level of plastocyanin providing direct evidence for the role of plastocyanin in noncyclic electron transport from water to NADP + . Phosphorylation cofactors and the uncoupler, NH 4 Cl, also cause shifts in the plastocyanin steady-state level in red light. Photosystem I light is able to photooxidize reduced plastocyanin, while Photosystem II light is only capable of photooxidation in the presence of inhibitors of the photoreduction. These results of studies on plastocyanin photoreactions in situ are discussed in terms of different proposed roles for plastocyanin in the chloroplast noncyclic electron transport chain.

The bound ferredoxin of chloroplasts: A role as the primary electron acceptor of photosystem I

Summary The bound ferredoxin of chloroplasts was found to be photoreduced at 25°K in whole chloroplasts as effectively in far-red light (λ > 700 nm) as in red light (λ

The orientation of the magnetic axes of the membrane-bound iron-sulfur clusters of spinach chloroplasts

Spinach chloroplast membranes were oriented onto mylar sheets by partial dehydration, and the orientation of the magnetic axes of membrane-bound paramagnetic clusters determined by electron paramagnetic resonance (EPR) spectroscopy. Our results indicate that the reduced Rieske iron-sulfur cluster signal is of orthorhombic symmetry oriented with th gy = 1.90 axis orthogonal to the membrane plane and with the gz = 2.03 axis in the membrane plane; the gx-axis is undetectable, presumably due to its broadness. If the Rieske center is a two-iron iron-sulfur cluster, we conclude that the iron-iron axis lies in the plane of the membrane. Illumination reduces the two bound chloroplast iron-sulfur proteins known as Clusters A and B. Center A is oriented such that gx = 1.86 and gy = 1.94 lie at an angle of about 40, and gz = 2.05 is at approximately 25, to the membrane plane. There are two possible orientations of Cluster B depending on the set of g-values assigned to this cluster. For one set of g-values, gz = 2.04 and gx = 1.89 are oriented in the plane of the membrane while gy = 1.92 is orthogonal to the plane. Alternatively, gz = 2.07 and gy = 1.94 are oriented approximately 50 and 40 to the membrane plane respectively, and gx = 1.80 is in the plane of the membrane. An additional light-induced signal at g = 2.15 oriented orthogonal to the plane is currently unexplained, as are other membrane perpendicular signals seen at g = 2.3 and g = 1.73 in dark-adapted samples.

Mössbauer Studies of Bonding in Iron Porphyrin–Ligand Systems

Mossbauer and electron paramagnetic resonance (EPR) spectra of several iron porphyrins have been obtained. The Mossbauer data for a series of high‐spin pentacoordinated iron (III) compounds establish that both the electric field gradient and the zero‐field splitting of the ionic Sz2 states follow the order fluoro<acetato<azido<chloro<bromo. The correlation of the two parameters can be qualitatively rationalized by considering the strong perturbing influence of the porphyrin ring on the relatively large high‐spin ion. The implications of the data for recent detailed theoretical models of the iron porphyrins are discussed. In pyridine, chloroprotohemin displays a Mossbauer spectrum with unusual temperature dependence and quadrupole splitting like that observed for acid methemoglobin. This suggests that these features may arise because of stronger interactions between orbitals of the more nearly planar metal ion with the porphyrin ring in hexacoordinated iron porphyrins. The isomer shifts of the Fe (II) dipy...