Evi Vinck

Analyzing heme proteins using EPR techniques: the heme-pocket structure of ferric mouse neuroglobin

In this work, an electron paramagnetic resonance (EPR) strategy to study the heme-pocket structure of low-spin ferric heme proteins is optimized. Frozen solutions of ferric mouse neuroglobin (mNgb) are analyzed by means of electron spin echo envelope modulation and pulsed electron–nuclear double resonance techniques. The hyperfine and nuclear quadrupole couplings of the directly coordinating heme and histidine nitrogens are derived and are discussed in comparison with known data of other ferric porphyrin compounds. In combination with the hyperfine matrices of the imidazole protons, the 14N EPR parameters reveal structural information on the heme pocket of mNgb that is in agreement with previous X-ray diffraction data on neuroglobins.

The Nerve Hemoglobin of the Bivalve Mollusc Spisula solidissima MOLECULAR CLONING, LIGAND BINDING STUDIES, AND PHYLOGENETIC ANALYSIS

Members of the hemoglobin (Hb) superfamily are present in nerve tissue of several vertebrate and invertebrate species. In vertebrates they display hexacoordinate heme iron atoms and are typically expressed at low levels (μm). Their function is still a matter of debate. In invertebrates they have a hexa- or pentacoordinate heme iron, are mostly expressed at high levels (mm), and have been suggested to have a myoglobin-like function. The native Hb of the surf clam, Spisula solidissima, composed of 162 amino acids, does not show specific deviations from the globin templates. UV-visible and resonance Raman spectroscopy demonstrate a hexacoordinate heme iron. Based on the sequence analogy, the histidine E7 is proposed as a sixth ligand. Kinetic and equilibrium measurements show a moderate oxygen affinity (P50 ∼0.6 torr) and no cooperativity. The histidine binding affinity is 100-fold lower than in neuroglobin. Phylogenetic analysis demonstrates a clustering of the S. solidissima nerve Hb with mollusc Hbs and myoglobins, but not with the vertebrate neuroglobins. We conclude that invertebrate nerve Hbs expressed at high levels are, despite the hexacoordinate nature of their heme iron, not essentially different from other intracellular Hbs. They most likely fulfill a myoglobin-like function and enhance oxygen supply to the neurons.

Characterization of a Globin-coupled Oxygen Sensor with a Gene-regulating Function

Globin-coupled sensors (GCSs) are multiple-domain transducers, consisting of a regulatory globin-like heme-binding domain and a linked transducer domain(s). GCSs have been described in both Archaea and bacteria. They are generally assumed to bind O2 (and perhaps other gaseous ligands) and to transmit a conformational change signal through the transducer domain in response to fluctuating O2 levels. In this study, the heme-binding domain, AvGReg178, and the full protein, AvGReg of the Azotobacter vinelandii GCS, were cloned, expressed, and purified. After purification, the heme iron of AvGReg178 was found to bind O2. This form was stable over many hours. In contrast, the predominant presence of a bis-histidine coordinate heme in ferric AvGReg was revealed. Differences in the heme pocket structure were also observed for the deoxygenated ferrous state of these proteins. The spectra showed that the deoxygenated ferrous derivatives of AvGReg178 and AvGReg are characterized by a penta-coordinate and hexa-coordinate heme iron, respectively. O2 binding isotherms indicate that AvGReg178 and AvGReg show a high affinity for O2 with P50 values at 20 °C of 0.04 and 0.15 torr, respectively. Kinetics of CO binding indicate that AvGReg178 carbonylation conforms to a monophasic process, comparable with that of myoglobin, whereas AvGReg carbonylation conforms to a three-phasic reaction, as observed for several proteins with bis-histidine heme iron coordination. Besides sensing ligands, in vitro data suggest that AvGReg(178) may have a role in O2-mediated NO-detoxification, yielding metAvGReg(178) and nitrate.

Analysing low-spin ferric complexes using pulse EPR techniques: a structure determination of bis (4-methylimidazole)(tetraphenylporphyrinato)iron(III)

Continuous-wave (CW) EPR has been extensively used as a characterization tool for low-spin ferric complexes. Using the bis(4-methylimidazole) complex of iron(III) tetraphenylporphyrin as an example we show how the combination of CW EPR and pulsed EPR techniques allows for a detailed structure analysis of such ferric complexes. Both proton HYSCORE and combination-peak experiments indicate that the imidazole ligand planes are (nearly) parallel to the gx axis (±20°). Simulations of the porphyrin nitrogen contributions to the HYSCORE spectra allow a determination of the g-axes frame in the molecular frame. Combination of these spectral results and the counter-rotation principle show that the gx and gy axes are rotated 5° (±5°) away from the Np–Fe–Np axes and that the imidazole ligand planes are counter-rotated over 5° (±5°). The nitrogen hyperfine and nuclear-quadrupole interactions of the pyrrole and imidazole nitrogens are determined and discussed as a function of the electronic structure.

Globin-Like Proteins in Caenorhabditis Elegans: In Vivo Localization, Ligand Binding and Structural Properties.

BackgroundThe genome of the nematode Caenorhabditis elegans contains more than 30 putative globin genes that all are transcribed. Although their translated amino acid sequences fit the globin fold, a variety of amino-acid substitutions and extensions generate a wide structural diversity among the putative globins. No information is available on the physicochemical properties and the in vivo expression.ResultsWe expressed the globins in a bacterial system, characterized the purified proteins by optical and resonance Raman spectroscopy, measured the kinetics and equilibria of O2 binding and determined the crystal structure of GLB-1* (CysGH2 → Ser mutant). Furthermore, we studied the expression patterns of glb-1 (ZK637.13) and glb-26 (T22C1.2) in the worms using green fluorescent protein technology and measured alterations of their transcript abundances under hypoxic conditions.GLB-1* displays the classical three-over-three α-helical sandwich of vertebrate globins, assembled in a homodimer associated through facing E- and F-helices. Within the heme pocket the dioxygen molecule is stabilized by a hydrogen bonded network including TyrB10 and GlnE7.GLB-1 exhibits high ligand affinity, which is, however, lower than in other globins with the same distal TyrB10-GlnE7 amino-acid pair. In the absence of external ligands, the heme ferrous iron of GLB-26 is strongly hexacoordinated with HisE7, which could explain its extremely low affinity for CO. This globin oxidizes instantly to the ferric form in the presence of oxygen and is therefore incapable of reversible oxygen binding.ConclusionThe presented data indicate that GLB-1 and GLB-26 belong to two functionally-different globin classes.

Discrimination of Geometrical Epoxide Isomers by ENDOR Spectroscopy and DFT Calculations: The Role of Hydrogen Bonds†

EPR and ENDOR spectroscopies combined with DFT calculations have revealed the selective binding of a cis over a trans epoxide to a chiral vanadyl salen complex (see picture). Complementary DFT calculations identified a weak electrostatic interaction supplemented by multiple hydrogen-bonding contacts as the origins of this selectivity. These observations were experimentally confirmed in frozen solution by orientation selective ENDOR measurements.

Studying High-Spin Ferric Heme Proteins by Pulsed EPR Spectroscopy: Analysis of the Ferric Form of the E7Q Mutant of Human Neuroglobin

In this work, the high-spin ferric form of the E7Q mutant of human neuroglobin (E7Q-NGB) is studied by X-band continuous-wave electron paramagnetic resonance (CW EPR) and hyperfine sublevel correlation (HYSCORE) spectroscopy. It is shown that the use of matched pulses in the HYSCORE experiment is essential to observe the nitrogen spectral contributions. The validity of approximating the high-spin Fe(III) system (S=5/2) as an effectiveS=1/2 system is tested and the consequences for the HYSCORE simulations are highlighted. Comparative HYSCORE experiments combined with deuterium exchange experiments for aquometmyoglobin and ferric E7Q-NGB clearly show that the heme iron of the latter protein is pentacoordinated, lacking the distal water. Furthermore, CW EPR experiments show that, at high pH, the E10K residue is coordinating to the heme iron in this globin. These observations are corroborated by resonance Raman experiments and could also be reproduced for other E7 mutants of human and mouse neuroglobin. Finally, the proton and nitrogen hyperfine and nuclear quadrupole parameters obtained for ferric E7Q-NGB are discussed in detail.