Betty J. Gaffney

The Paramagnetic Resonance Spectra of Spin Labels in Phospholipid Membranes

Abstract An analysis of the paramagnetic resonance spectra of phospholipid spin labels incorporated in phospholipid bilayer membranes is described. The analysis takes into account rapid anisotropic motion which gives rise to an effective spin Hamiltonian. The broadening of transitions between the eigenstates of this effective Hamiltonian is approximated using Redfield relaxation theory. The analysis of the spectra also takes into account spacial distributions of the axes of the effective spin Hamiltonian as well as a field-induced orientation of the lipid molecules. Resonance spectra are reported for isotropic samples, as well as oriented multibilayers (smectic liquid crystals), at 9.3 and 35.0 GHz, for various orientations of the applied field. Order parameters for the phospholipid spin labels are determined, and calculations are made of order parameters appropriate to different types of spectroscopic time-averaging, including ensemble-average order parameters that correspond to long-time averages.

Structure and mechanism of lipoxygenases

In mammals, lipoxygenases catalyze the formation of hydroperoxides as the first step in the biosynthesis of several inflammatory mediators. The substrate of this reaction, arachidonic acid, is the key precursor of two families of potent physiological effectors. It is the branch point between two central pathways: one, involving the enzyme cyclooxygenase, leads to the synthesis of prostaglandins and thromboxanes; the other, involving lipoxygenases, leads to the synthesis of leukotrienes and lipoxins, compounds that regulate important cellular responses in inflammation and immunity. While aspirin and other non-steroidal anti-inflammatory compounds are potent inhibitors of cyclooxygenase, no effective pharmacological inhibitor of lipoxygenase is presently available. Lipoxygenases are large non-heme, iron-containing enzymes that use molecular oxygen for the diooxygenation of arachidonic acid to form hydroperoxides, the first step in the biosynthetic pathways leading to leukotrienes and lipoxins. Because of the importance of these compounds, lipoxygenases have been the subject of extensive study: from detailed kinetic measurements to cloning, expression, and site-directed mutagenesis. The sequences of over 50 lipoxygenases have been reported. In addition, the structure of soybean lipoxygenase-1, determined by X-ray diffraction methods, has recently been reported. The structure revealed that the 839 amino acids in the protein are organized in two domains: a beta-sheet N-terminal domain and a large, mostly helical C-terminal domain. The iron is present in the C-terminal domain facing two internal cavities that are probably the conduits through which the fatty acid and molecular oxygen gain access to the metal. Models of the mammalian lipoxygenases based on the soybean structure provide clues about the structural determinants of the positional specificity of the enzyme, and can be used as targets for the design of more effective inhibitors.

Effect of the Viral Proteins on the Fluidity of the Membrane Lipids in Sindbis Virus

Abstract The fluidity of the lipids in the membrane of Sindbis virus was studied by electron paramagnetic resonance spectroscopy. The lipids in the viral membrane are noticeably less fluid than the lipids in the membrane of the cells in which the virus was grown. This difference in fluidity is not due simply to differences in lipid composition but instead appears to be the result of the interaction of the viral proteins with the membrane lipids. This conclusion seems clear because (i) the viral lipids are more fluid after extraction with chloroform/methanol than in the lipid bilayer of the virion and because (ii) the viral membrane is made more fluid by proteolytic digestion of the viral glycoproteins. The possible role of this viral protein-mediated alteration of the physical state of membrane lipids in the maturation of Sindbis virions is discussed.

Structure Conservation in Lipoxygenases: Structural Analysis of Soybean Lipoxygenase-1 and Modeling of Human Lipoxygenases

Lipoxygenases are a class of non‐heme iron dioxygenases which catalyze the hydroperoxidation of fatty acids for the biosynthesis of leukotrienes and lipoxins. The structure of the 839‐residue soybean lipoxygenase‐1 was used as a template to model human 5‐, 12‐, and 15‐lipoxygenases. A distance‐based algorithm for placing side chains in a low homology environment (only the four iron ligands were fixed during side chain placement) was devised. Twenty‐six of the 56 conserved lipoxygenase residues were grouped in four distinct regions of the enzyme. These regions were analyzed to discern whether the side chain interactions could be duplicated in the models or whether alternate conformers should be considered. The effects of site directed mutagenesis variants were rationalized using the models of the human lipoxygenases. In particular, variants which shifted positional specificity between 12‐ and 15‐lipoxygenase activity were analyzed. Analysis of active site residues produced a model which accounts for observed lipoxygenase positional specificity and stereospecificity.

On the Relationships of Substrate Orientation, Hydrogen Abstraction, and Product Stereochemistry in Single and Double Dioxygenations by Soybean Lipoxygenase-1 and Its Ala542Gly Mutant

Recent findings associate the control of stereochemistry in lipoxygenase (LOX) catalysis with a conserved active site alanine for S configuration hydroperoxide products, or a corresponding glycine for R stereoconfiguration. To further elucidate the mechanistic basis for this stereocontrol we compared the stereoselectivity of the initiating hydrogen abstraction in soybean LOX-1 and an Ala542Gly mutant that converts linoleic acid to both 13S and 9R configuration hydroperoxide products. Using 11R-3H- and 11S-3H-labeled linoleic acid substrates to examine the initial hydrogen abstraction, we found that all the primary hydroperoxide products were formed with an identical and highly stereoselective pro-S hydrogen abstraction from C-11 of the substrate (97-99% pro-S-selective). This strongly suggests that 9R and 13S oxygenations occur with the same binding orientation of substrate in the active site, and as the equivalent 9R and 13S products were formed from a bulky ester derivative (1-palmitoyl-2-linoleoylphosphatidylcholine), one can infer that the orientation is tail-first. Both the EPR spectrum and the reaction kinetics were altered by the R product-inducing Ala-Gly mutation, indicating a substantial influence of this Ala-Gly substitution extending to the environment of the active site iron. To examine also the reversed orientation of substrate binding, we studied oxygenation of the 15S-hydroperoxide of arachidonic acid by the Ala542Gly mutant soybean LOX-1. In addition to the usual 5S, 15S- and 8S, 15S-dihydroperoxides, a new product was formed and identified by high-performance liquid chromatography, UV, gas chromatography-mass spectrometry, and NMR as 9R, 15S-dihydroperoxyeicosa-5Z,7E,11Z,13E-tetraenoic acid, the R configuration “partner” of the normal 5S,15S product. This provides evidence that both tail-first and carboxylate end-first binding of substrate can be associated with S or R partnerships in product formation in the same active site.

Determination of relative spin concentration in some high-spin ferric proteins using E/D-distribution in electron paramagnetic resonance simulations

Lineshape simulations are presented for the multiple, overlapping X-band electron paramagnetic resonance (EPR) spectra in two non-heme, high-spin iron proteins: phenylalanine hydroxylase (PAH) and diferric transferrin. The aim of the calculations is to determine the fraction of iron contributing to each of the sites visible by EPR. The simulations are limited to the experimentally accessible transitions occurring at g-values greater than 1.7. In both PAH and transferrin, at least one of the iron sites is characterized by the ratio of zero-field splitting parameters, E/D, near 1/3 and a broad, asymmetric lineshape. A distribution in E/D-values is used in the simulations to account for this breadth and asymmetry. To test the E/D-distribution model, experimental X-band spectra of diferric transferrin at several salt concentrations are fit by simulation. In this test, first the low-field features arising from transitions between the lowest Kramers doublet levels are simulated using E/D-distributions for two sites. Second, parameters that provide a good fit for the lowest doublet transitions are shown also to fit the resonance near an effective g-value of 4.3 from the middle Kramers doublet transition. When applied to spectra of PAH in the resting state, the E/D-distribution approach accounts for the intensity of one of the two major species of iron. The other species is characterized by E/D = 0.032, and the spectrum of this portion of the resting enzyme may be simulated using a frequency-swept Gaussian lineshape. Spectra for the enzyme in an inhibitor-saturated state are also simulated. The simulations are consistent with previous biochemical studies that indicate that only the E/D = 0.032 form of iron participates in catalysis.

Crystal structures of vegetative soybean lipoxygenase VLX-B and VLX-D, and comparisons with seed isoforms LOX-1 and LOX-3.

The lipoxygenase family of lipid‐peroxidizing, nonheme iron dioxygenases form products that are precursors for diverse physiological processes in both plants and animals. In soybean (Glycine max), five vegetative isoforms, VLX‐A, VLX‐B, VLX‐C, VLX‐D, VLX‐E, and four seed isoforms LOX‐1, LOX‐2, LOX‐3a, LOX‐3b have been identified. In this study, we determined the crystal structures of the substrate‐free forms of two major vegetative isoforms, with distinct enzymatic characteristics, VLX‐B and VLX‐D. Their structures are similar to the two seed isoforms, LOX‐1 and LOX‐3, having two domains with similar secondary structural elements: a β‐barrel N‐terminal domain containing highly flexible loops and an α‐helix‐rich C‐terminal catalytic domain. Detailed comparison of the structures of these two vegetative isoforms with the structures of LOX‐1 and LOX‐3 reveals important differences that help explain distinct aspects of the activity and positional specificity of these enzymes. In particular, the shape of the three branches of the internal subcavity, corresponding to substrate‐binding and O2 access, differs among the isoforms in a manner that reflects the differences in positional specificities. Proteins 2006.

Structure and interaction with phospholipids of a prokaryotic lipoxygenase from Pseudomonas aeruginosa

Lipoxygenases (LOXs), which are essential in eukaryotes, have no confirmed function in prokaryotes that are devoid of polyunsaturated fatty acids. The structure of a secretable LOX from Pseudomonas aeruginosa (Pa_LOX), the first available from a prokaryote, presents significant differences with respect to eukaryotic LOXs, including a cluster of helices acting as a lid to the active center. The mobility of the lid and the structural variability of the N‐terminal region of Pa_LOX was confirmed by comparing 2 crystal forms. The binding pocket contains a phosphatidylethanolamine phospholipid with branches of 18 (sn‐1) and 14/16 (sn‐2) carbon atoms in length. Carbon atoms from the sn‐1 chain approach the catalytic iron in a manner that sheds light on how the enzymatic reaction might proceed. The findings in these studies suggest that Pa_LOX has the capacity to extract and modify unsaturated phospholipids from eukaryotic membranes, allowing this LOX to play a role in the interaction of P. aeruginosa with host cells.—Garreta, A., Val‐Moraes, S. P., García‐Fernández, Q., Montserrat Busquets, C. J., Oliver, A., Ortiz, A., Gaffney, B. J., Fita, I., Manresa, A., Carpena, X., Structure and interaction with phospholipids of a prokaryotic lipoxygenase from Pseudomonas aeruginosa. FASEB J. 27, 4811‐4821 (2013). www.fasebj.org

Simulation of the EMR Spectra of High-Spin Iron in Proteins

Very detailed information about the energy levels and orientations of d orbitals in heme proteins has been obtained by combining EMR studies of paramagnetic samples with other structural information from X-ray crystallography and optical studies. As a result, the chemistry of heme enzymes can be discussed in detail. While the aim of this chapter is to review progress in bringing the chemistry of mononuclear iron centers in nonheme proteins to a similar level of knowledge, our understanding of line shape analysis for high-spin iron is dominated by the vast literature on heme samples. We begin this introduction with some of the history of EMR spectroscopy of methemoglobin and metmyoglobin.

Locating a lipid at the portal to the lipoxygenase active site.

Lipoxygenase enzymes initiate diverse signaling pathways by specifically directing oxygen to different carbons of arachidonate and other polyunsaturated acyl chains, but structural origins of this specificity have remained unclear. We therefore determined the nature of the lipoxygenase interaction with the polar-end of a paramagnetic lipid by electron paramagnetic resonance spectroscopy. Distances between selected grid points on soybean seed lipoxygenase-1 (SBL1) and a lysolecithin spin-labeled on choline were measured by pulsed (electron) dipolar spectroscopy. The protein grid was designed by structure-based modeling so that five natural side chains were replaced with spin labels. Pairwise distances in 10 doubly spin-labeled mutants were examined by pulsed dipolar spectroscopy, and a fit to the model was optimized. Finally, experimental distances between the lysolecithin spin and each single spin site on SBL1 were also obtained. With these 15 distances, distance geometry localized the polar-end and the spin of the lysolecithin to the region between the two domains in the SBL1 structure, nearest to E236, K260, Q264, and Q544. Mutation of a nearby residue, E256A, relieved the high pH requirement for enzyme activity of SBL1 and allowed lipid binding at pH 7.2. This general approach could be used to locate other flexible molecules in macromolecular complexes.