Electron-nuclear double resonance spectroscopy of 15N-enriched phthalate dioxygenase from Pseudomonas cepacia proves that two histidines are coordinated to the [2Fe-2S] Rieske-type clusters

We have performed ENDOR spectroscopy at microwave frequencies of 9 and 35 GHz at 2 K on the reduced Rieske-type [2Fe-2S] cluster of phthalate dioxygenase (PDO) from Pseudomonas cepacia. Four samples have been examined: (1) 14N (natural abundance); (2) uniformly 15N labeled; (3) [15N]histidine in a 14N background; (4) [14N]histidine in a 15N background. These studies establish unambiguously that two of the ligands to the Rieske [2Fe-2S] center are nitrogens from histidine residues. This contrasts with classical ferredoxin-type [2Fe-2S] centers in which all ligation is by sulfur of cysteine residues. Analysis of the polycrystalline ENDOR patterns has permitted us to determine for each nitrogen ligand the principal values of the hyperfine tensor and its orientation with respect to the g tensor, as well as the 14N quadrupole coupling tensor. The combination of these results with earlier M?ssbauer and resonance Raman studies supports a model for the reduced cluster with both histidyl ligands bound to the ferrous ion of the spin-coupled [Fe2+ (S = 2), Fe3+ (S = 5/2)] pair. The analyses of 15N hyperfine and 14N quadrupole coupling tensors indicate that the geometry of ligation at Fe2+ is approximately tetrahedral, with the (Fe)2(N)2 plane corresponding to the g1-g3 plane, and that the planes of the histidyl imidazoles lie near that plane, although they could not both lie in the plane. The bonding parameters of the coordinated nitrogens are fully consistent with those of an spn hybrid on a histidyl nitrogen coordinated to Fe. Differences in 14N ENDOR line width provide evidence for different mobilities of the two imidazoles when the protein is in fluid solution. We conclude that the structure deduced here for the PDO cluster is generally applicable to the full class of Rieske-type centers.     

Potentiometric study of cytochrome c1aa3 from Thermus thermophilus

We have examined the redox behavior of the cytochrome c1aa3 complex from Thermus thermophilus. In potentiometric titrations the cytochrome c behaves as an independent center having n = 1 and E = 205 mV (NHE). Under the assumption that the individual centers equilibrate independently in this experiment, changes in the absorption band at 603 nm have been resolved into two components: cytochrome a (n = 1, Em = 270 mV, 60% spectral contribution) and cytochrome a3 (n = 2, Em = 360 mV, 40% spectral contribution). The n = 2 process was attributed to strong chemical coupling between cytochrome a3 and CuB. The enzyme was also titrated with a mixture of NADH and PMS, and the results are shown not to conform to a model of intramolecular equilibrium according to the equilibrium constants obtained from the potentiometric titration. It is suggested that a conformational equilibrium within the complex may control electron transfer between cytochromes a and a3.     

Studies of the ferredoxin from Thermus thermophilus

The soluble ferredoxin from Thermus thermophilus was examined by M?ssbauer and EPR spectroscopies and by reductive titrations. These studies demonstrate the presence of one 3Fe center, responsible for the characteristic g = 2.02 EPR signal in the oxidized protein, and one [4Fe-4S] center which is responsible for the rhombic EPR spectrum of the fully reduced protein. These assignments should replace those made by Ohnishi et al. (Ohnishi, T., Blum, H., Sato, S., Nakazawa, K., Hon-nami, K., and Oshima, T. (1980) J. Biol. Chem. 255, 345-348) prior to the discovery of the 3Fe clusters. The amino acid composition was determined and is discussed with reference to recent structural studies of 7Fe ferredoxins.     

Probing protein-cofactor interactions in the terminal oxidases by second derivative spectroscopy: study of bacterial enzymes with cofactor substitutions and heme A model compounds.

Second derivative absorption spectra are reported for the aa3-cytochrome c oxidase from bovine cardiac mitochondria, the aa3-600 ubiquinol oxidase from Bacillus subtilis, the ba3-cytochrome c oxidase from Thermus thermophilis, and the aco-cytochrome c oxidase from Bacillus YN-2000. Together these enzymes provide a range of cofactor combinations that allow us to unequivocally identify the origin of the 450-nm absorption band of the terminal oxidases as the 6-coordinate low-spin heme, cytochrome a. The spectrum of the aco-cytochrome c oxidase further establishes that the split Soret band of cytochrome a, with features at 443 and 450 nm, is common to all forms of the enzyme containing ferrocytochrome a and does not depend on ligand occupancy at the other heme cofactor as previously suggested. To test the universality of this Soret band splitting for 6-coordinate low-spin heme A systems, we have reconstituted purified heme A with the apo forms of the heme binding proteins, hemopexin, histidine-proline-rich glycoprotein and the H64V/V68H double mutant of human myoglobin. All 3 proteins bound the heme A as a (bis)histidine complex, as judged by optical and resonance Raman spectroscopy. In the ferroheme A forms, none of these proteins displayed evidence of Soret band splitting. Heme A-(bis)imidazole in aqueous detergent solution likewise failed to display Soret band splitting. When the cyanide-inhibited mixed-valence form of the bovine enzyme was partially denatured by chemical or thermal means, the split Soret transition of cytochrome a collapsed into a single band at 443 nm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Seagrass ecosystem multifunctionality under the rise of a flagship marine megaherbivore

Large grazers (megaherbivores) have a profound impact on ecosystem functioning. However, how ecosystem multifunctionality is affected by changes in megaherbivore populations remains poorly understood. Understanding the total impact on ecosystem multifunctionality requires an integrative ecosystem approach, which is especially challenging to obtain in marine systems. We assessed the effects of experimentally simulated grazing intensity scenarios on ecosystem functions and multifunctionality in a tropical Caribbean seagrass ecosystem. As a model, we selected a key marine megaherbivore, the green turtle, whose ecological role is rapidly unfolding in numerous foraging areas where populations are recovering through conservation after centuries of decline, with an increase in recorded overgrazing episodes. To quantify the effects, we employed a novel integrated index of seagrass ecosystem multifunctionality based upon multiple, well-recognized measures of seagrass ecosystem functions that reflect ecosystem services. Experiments revealed that intermediate turtle grazing resulted in the highest rates of nutrient cycling and carbon storage, while sediment stabilization, decomposition rates, epifauna richness, and fish biomass are highest in the absence of turtle grazing. In contrast, intense grazing resulted in disproportionally large effects on ecosystem functions and a collapse of multifunctionality. These results imply that (i) the return of a megaherbivore can exert strong effects on coastal ecosystem functions and multifunctionality, (ii) conservation efforts that are skewed toward megaherbivores, but ignore their key drivers like predators or habitat, will likely result in overgrazing-induced loss of multifunctionality, and (iii) the multifunctionality index shows great potential as a quantitative tool to assess ecosystem performance. Considerable and rapid alterations in megaherbivore abundance (both through extinction and conservation) cause an imbalance in ecosystem functioning and substantially alter or even compromise ecosystem services that help to negate global change effects. An integrative ecosystem approach in environmental management is urgently required to protect and enhance ecosystem multifunctionality.     

An Ustilago maydis Mutant Partially Blocked in P45014DM Activity Is Hypersensitive to Azole Fungicides

An Ustilago maydis ergosterol biosynthesis mutant (A14) which is partially blocked in sterol 14alpha-demethylase (P45014DM) activity is described. This mutant accumulated the abnormal 14alpha-methyl sterols, eburicol, 14alpha-methylfecosterol, and obtusifoliol, along with significant amounts of ergosterol. Although the A14 mutant grew nearly as well as the wild type, it was impaired in cell extension growth, which indicated a dysfunction in apical cell wall synthesis. The mutant was also found to be hypersensitive to the azole fungicides penconazole and tebuconazole.     

The relative effectiveness of three techniques to induce the trophotropic response

The purpose of this study was to examine the relative effectiveness of electromyographic biofeedback training(EMG BFT), remeditation, and progressive muscle relaxation(PMR) in eliciting a relaxation or trophotropic response as measured by frontalis muscle tension, heart rate, electrodermal response, respiration rate, and skin temperature. Fifty-four college students were randomly assigned to one of five groups:(1) control,(2) placebo control,(3) EMG BFT,(4) meditation,(5) PMR. After baseline measures were obtained subjects were trained in 10 30-minute training sessions and posttested. Comparisons by ANOVAs indicated there was a significant decrease in muscle tension in the EMG BFT and meditation groups and significant decreases in respiration rate in the meditation and PMR groups. No other changes were attributed to treatment.     

An investigation of Chromatium vinosum high-potential iron-sulfur 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.