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.

The second helix in lipoxygenases adapts to permit substrate access to the active site, but details of this process are varied and poorly understood. We therefore examined the dynamics of helix 2 in solutions of spin-labeled soybean lipoxygenase-1 and spin relaxation at 60 K of the spin-labels by catalytic iron. Helix 2 in soybean lipoxygenase structures is surface-exposed and contains one turn of π-helix, centrally located. A site-directed spin-label scan of 18 of the 21 helix 2 residues, and electron paramagnetic resonance, showed that the π-helical segment became unusually mobile, on a nanosecond time scale, under conditions favoring substrate binding (pH 9 and lipid addition), while segments before and after had relatively unchanged dynamics. Backbone dynamics of residues in the π-helical segment appeared to be correlated, at pH 9. Samples also were frozen to examine the polarity and proticity of the local environments, the effect of the local environment on intrinsic relaxation, and dipolar relaxation by two symmetries of catalytic iron. The average hyperfine tensor component, Azz, of four π-helix residues decreased by 1.75 G, with an increase in pH from 7 to 9, while it remained unaffected for nearby buried residues. Power saturation data suggested the change in polarity specific to the π-helix altered the intrinsic relaxation rates. Different symmetries of iron contributed to distance-dependent magnetic relaxation. We interpret these data to mean that a π-helix in the second helix of plant lipoxygenases is highly dynamic and is the site where lipid chains penetrate to inner helices that outline the substrate pocket.

openAccessArticle: TruePage Range: 63a-63adoi: 10.1016/j.bpj.2011.11.376Harvest Date: 2016-01-12 15:08:09issueName:cover date: 2012-01-31pubType

A persistent puzzle about lipoxygenase enzymes is how specificity for position and stereochemistry of oxygen addition to polyunsaturated fatty acids is achieved. In lipoxygenase structures, a curved substrate channel approaches the centrally located active site, with ends of the curve near different surface locations. A long‐standing suggestion is that varied specificity results from reversed placement of the polar and methyl ends of the substrate within highly similar protein structures. We approach this problem by placing a spin label on the polar end of a lipoxygenase substrate analog and use paramagnetic distance geometry to locate the substrate spin relative to selected protein positions. Soybean seed lipoxygenase‐1 has been substituted with spin labels at five sites. A five‐point representation of the structure is obtained by measuring ten inter‐site distances by pulsed dipolar EPR spectroscopy (PDS) in doubly spin labeled enzyme constructs. Refinement of spin label coordinates results from modeling and fits to the native protein structure. Finally, the substrate analog polar end is found with five more distance measurements between substrate and protein spins. The spin of the substrate analog is just outside the conjunction of helices‐2 and ‐11. Detailed spin labeling of helix‐2 reveals flexibility permitting entrance to the internal cavity. 1NIH GM65268 and 2NIH RR016292