Inés García-Rubio

Development of cellular magnetic dipoles in magnetotactic bacteria.

Magnetotactic bacteria benefit from their ability to form cellular magnetic dipoles by assembling stable single-domain ferromagnetic particles in chains as a means to navigate along Earths magnetic field lines on their way to favorable habitats. We studied the assembly of nanosized membrane-encapsulated magnetite particles (magnetosomes) by ferromagnetic resonance spectroscopy using Magnetospirillum gryphiswaldense cultured in a time-resolved experimental setting. The spectroscopic data show that 1), magnetic particle growth is not synchronized; 2), the increase in particle numbers is insufficient to build up cellular magnetic dipoles; and 3), dipoles of assembled magnetosome blocks occur when the first magnetite particles reach a stable single-domain state. These stable single-domain particles can act as magnetic docks to stabilize the remaining and/or newly nucleated superparamagnetic particles in their adjacencies. We postulate that docking is a key mechanism for building the functional cellular magnetic dipole, which in turn is required for magnetotaxis in bacteria.

Probing the role of the proximal heme ligand in cytochrome P450cam by recombinant incorporation of selenocysteine

The unique monooxygenase activity of cytochrome P450cam has been attributed to coordination of a cysteine thiolate to the heme cofactor. To investigate this interaction, we replaced cysteine with the more electron-donating selenocysteine. Good yields of the selenoenzyme were obtained by bacterial expression of an engineered gene containing the requisite UGA codon for selenocysteine and a simplified yet functional selenocysteine insertion sequence (SECIS). The sulfur-to-selenium substitution subtly modulates the structural, electronic, and catalytic properties of the enzyme. Catalytic activity decreases only 2-fold, whereas substrate oxidation becomes partially uncoupled from electron transfer, implying a more complex role for the axial ligand than generally assumed.

Metal Dependence of Oxalate Decarboxylase Activity

Bacillus subtilis oxalate decarboxylase (OxDC) catalyzes the conversion of oxalate into CO(2) and formate. The enzyme is composed of two cupin domains, each of which contains a Mn(II) ion. Although there is general agreement that Mn(II) in the N-terminal domain mediates OxDC-catalyzed decarboxylation, legitimate questions have been raised concerning the function (if any) of the Mn(II) bound in the C-terminal cupin domain. We have investigated this problem using a series of OxDC mutants in which Mn(II) binding is perturbed by mutagenesis of Glu-101 and Glu-280, which coordinate the metal in the N-terminal and C-terminal domains, respectively. We now demonstrate that decarboxylase activity and total manganese content are sensitive to modifications in either metal-binding glutamate residue. These findings, in combination with EPR measurements, raise the possibility that the C-terminal Mn(II) center can catalyze the decarboxylation reaction. Further support for this conclusion has been provided from a combination of in vivo and in vitro strategies for preparing wild-type OxDC in which Mn(II) is incorporated to a variety of extents. Kinetic characterization of these variants shows that OxDC activity is linearly correlated with manganese content, as might be expected if both sites can catalyze the breakdown of oxalate into formate and CO(2). These studies also represent the first unequivocal demonstration that OxDC activity is uniquely mediated by manganese.

A multi-frequency pulse EPR and ENDOR approach to study strongly coupled nuclei in frozen solutions of high-spin ferric heme proteins.

In spite of the tremendous progress in the field of pulse electron paramagnetic resonance (EPR) in recent years, these techniques have been scarcely used to investigate high-spin (HS) ferric heme proteins. Several technical and spin-system-specific reasons can be identified for this. Additional problems arise when no single crystals of the heme protein are available. In this work, we use the example of a frozen solution of aquometmyoglobin (metMb) to show how a multi-frequency pulse EPR approach can overcome these problems. In particular, the performance of the following pulse EPR techniques are tested: Davies electron nuclear double resonance (ENDOR), hyperfine correlated ENDOR (HYEND), electron-electron double resonance (ELDOR)-detected NMR, and several variants of hyperfine sublevel correlation (HYSCORE) spectroscopy including matched and SMART HYSCORE. The pulse EPR experiments are performed at X-, Q- and W-band microwave frequencies. The advantages and drawbacks of the different methods are discussed in relation to the nuclear interaction that they intend to reveal. The analysis of the spectra is supported by several simulation procedures, which are discussed. This work focuses on the analysis of the hyperfine and nuclear-quadrupole tensors of the strongly coupled nuclei of the first coordination sphere, namely, the directly coordinating heme and histidine nitrogens and the 17O nucleus of the distal water ligand. For the latter, 17O-isotope labeling was used. The accuracy of our results and the spectral resolution are compared in detail to an earlier single-crystal continuous-wave ENDOR study on metMb, and it will be shown how additional information can be obtained from the multi-frequency approach. The current work is therefore prone to become a template for future EPR/ENDOR investigations of HS ferric heme proteins for which no single crystals are available.

Haptoglobin Preserves Vascular Nitric Oxide Signaling during Hemolysis

RATIONALE Hemolysis occurs not only in conditions such as sickle cell disease and malaria but also during transfusion of stored blood, extracorporeal circulation, and sepsis. Cell-free Hb depletes nitric oxide (NO) in the vasculature, causing vasoconstriction and eventually cardiovascular complications. We hypothesize that Hb-binding proteins may preserve vascular NO signaling during hemolysis. OBJECTIVES Characterization of an archetypical function by which Hb scavenger proteins could preserve NO signaling during hemolysis. METHODS We investigated NO reaction kinetics, effects on arterial NO signaling, and tissue distribution of cell-free Hb and its scavenger protein complexes. MEASUREMENTS AND MAIN RESULTS Extravascular translocation of cell-free Hb into interstitial spaces, including the vascular smooth muscle cell layer of rat and pig coronary arteries, promotes vascular NO resistance. This critical disease process is blocked by haptoglobin. Haptoglobin does not change NO dioxygenation rates of Hb; rather, the large size of the Hb:haptoglobin complex prevents Hb extravasation, which uncouples NO/Hb interaction and vasoconstriction. Size-selective compartmentalization of Hb functions as a substitute for red blood cells after hemolysis and preserves NO signaling in the vasculature. We found that evolutionarily and structurally unrelated Hb-binding proteins, such as PIT54 found in avian species, functionally converged with haptoglobin to protect NO signaling by sequestering cell-free Hb in large protein complexes. CONCLUSIONS Sequential compartmentalization of Hb by erythrocytes and scavenger protein complexes is an archetypical mechanism, which may have supported coevolution of hemolysis and normal vascular function. Therapeutic supplementation of Hb scavengers may restore vascular NO signaling and attenuate disease complications in patients with hemolysis.

Cryogenic 35GHz pulse ENDOR probehead accommodating large sample sizes: Performance and applications.

The construction and performance of a cryogenic 35GHz pulse electron nuclear double resonance (ENDOR) probehead for large samples is presented. The resonator is based on a rectangular TE(102) cavity in which the radio frequency (rf) B(2)-field is generated by a two turn saddle ENDOR coil crossing the resonator along the sample axis with minimal distance to the sample tube. An rf power efficiency factor is used to define the B(2)-field strength per square-root of the transmitted rf power over the frequency range 2-180MHz. The distributions of the microwave B(1)- and E(1)-field, and the rf B(2)-field are investigated by electromagnetic field calculations. All dielectrics, the sample tube, and coupling elements are included in the calculations. The application range of the probehead and the advantages of using large sample sizes are demonstrated and discussed on a number of paramagnetic samples containing transition metal ions.

Evolution of magnetic anisotropy and thermal stability during nanocrystal-chain growth

We compare measurements and simulations of ferromagnetic resonance spectra of magnetite nanocrystal-chains at different growth-stages. By fitting the spectra, we extracted the cubic magnetocrystalline anisotropy field and the uniaxial dipole field at each stage. During the growth of the nanoparticle-chain assembly, the magnetocrystalline anisotropy grows linearly with increasing particle diameter. Above a threshold average diameter of D ≈ 23 nm, a dipole field is generated, which then increases with particle size and the ensemble becomes thermally stable. These findings demonstrate the anisotropy evolution on going from nano to mesoscopic scales and the dominance of dipole fields over crystalline fields in one-dimensional assemblies.

Combination of X-ray crystallography, SAXS and DEER to obtain the structure of the FnIII-3,4 domains of integrin α6β4

The structure of the FnIII-3,4 region of integrin β4 was solved using a hybrid approach that combines crystallographic structures, SAXS, DEER and molecular modelling. The structure helps in understanding how integrin β4 might bind to other hemidesmosomal proteins and mediate signalling.

Magnetic anisotropy of non-interacting collinear nanocrystal-chains

The magnetic anisotropy of linear chains of spherical magnetite nanocrystals was investigated by means of angle-resolved ferromagnetic resonance spectroscopy, in order to determine the different anisotropy contributions. The linear assembly of nanocrystals generates an interaction-induced uniaxial anisotropy, which is nearly an order of magnitude stronger than the intrinsic magnetocrystalline anisotropy of magnetite, and can only exist in magnetic nano-chains, where the easy axes of the nanocrystals are collinear.

Spin trapping combined with quantitative mass spectrometry defines free radical redistribution within the oxidized hemoglobin:haptoglobin complex.

Extracellular or free hemoglobin (Hb) accumulates during hemolysis, tissue damage, and inflammation. Heme-triggered oxidative reactions can lead to diverse structural modifications of lipids and proteins, which contribute to the propagation of tissue damage. One important target of Hb׳s peroxidase reactivity is its own globin structure. Amino acid oxidation and crosslinking events destabilize the protein and ultimately cause accumulation of proinflammatory and cytotoxic Hb degradation products. The Hb scavenger haptoglobin (Hp) attenuates oxidation-induced Hb degradation. In this study we show that in the presence of hydrogen peroxide (H2O2), Hb and the Hb:Hp complex share comparable peroxidative reactivity and free radical generation. While oxidation of both free Hb and Hb:Hp complex generates a common tyrosine-based free radical, the spin-trapping reaction with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) yields dissimilar paramagnetic products in Hb and Hb:Hp, suggesting that radicals are differently redistributed within the complex before reacting with the spin trap. With LC-MS(2) mass spectrometry we assigned multiple known and novel DMPO adduct sites. Quantification of these adducts suggested that the Hb:Hp complex formation causes extensive delocalization of accessible free radicals with drastic reduction of the major tryptophan and cysteine modifications in the β-globin chain of the Hb:Hp complex, including decreased βCys93 DMPO adduction. In contrast, the quantitative changes in DMPO adduct formation on Hb:Hp complex formation were less pronounced in the Hb α-globin chain. In contrast to earlier speculations, we found no evidence that free Hb radicals are delocalized to the Hp chain of the complex. The observation that Hb:Hp complex formation alters free radical distribution in Hb may help to better understand the structural basis for Hp as an antioxidant protein.