Fluctuations of an Exposed π-Helix Involved in Lipoxygenase Substrate Recognition

Abstract

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.

Site‐directed spin label electron paramagnetic resonance spectroscopy as a probe of conformational dynamics in the Fe(III) “locked‐off” state of the CO‐sensing transcription factor CooA

“…The size of CooA (50 kDa) and presence of a paramagnetic heme in the Fe(III) “ locked‐off ” state render such NMR experiments challenging; however, these constraints do not limit the use of site‐specifically incorporated nitroxide spin labels that report on local protein dynamics on the μs‐ms timescale . SDSL‐EPR spectroscopy, which has been utilized to study conformational exchange and structural transitions in a number of soluble and membrane‐bound proteins, is well‐suited to probe conformational dynamics in CooA . In this study, we compare conformational dynamics of five cysteine substitution variants of Rr CooA in the Fe(III)“ locked‐off ” state using two nitroxide spin labels: MTSL (2,2,5,5‐tetramethyl‐1‐oxyl‐3‐methyl methanethiosulfonate spin label) and MAL‐6 (N‐(2,2,6,6‐tetramethylpiperidin‐4‐yl‐1‐oxyl)maleimide) (Fig.…”

“…[40][41][42][43] SDSL-EPR spectroscopy, which has been utilized to study conformational exchange and structural transitions in a number of soluble and membrane-bound proteins, is well-suited to probe conformational dynamics in CooA. [44][45][46][47][48] In this study, we compare conformational dynamics of five cysteine substitution variants of Rr CooA in the Fe(III)"locked-off" state using two nitroxide spin labels: MTSL (2,2,5,5-tetramethyl-1-oxyl-3-methyl methanethiosulfonate spin label) and MAL-6 (N-(2,2,6,6-tetramethylpiperidin-4-yl-1-oxyl)maleimide) ( Fig. 2).…”

The structural basis for specificity in lipoxygenase catalysis

“…Their results, which allow them to observe mobility along the helix at a time scale relevant to catalysis and in the presence of substrate, put the "portal" just a few residues up from Thr-259 at a conserved p-helical segment of a2 at 261-267. 37 Earlier work by the investigators led them to position the polar end of the fatty acid at the "entrance" end of the cavity. 38 Their data suggest a model in which the hydrocarbon region of the substrate interacts with the p-helix segment to grease its way between the two helices (a2 and the arched helix) that shield the active site.…”

EPR Spectroscopic Studies of Lipoxygenases

“…[36,39] To examineS BL1 in solution at ambient temperature, a series was prepared of spin labeled derivatives of the cysteinefree enzyme,i nw hich each of eighteena mino acids in helix 2 was replaced with cysteinea nd then spin labeled (SDSL). [52] The effects of pH variation from 7t o9a nd of lipid addition on the room temperature EPR spectra were examined. In this case, the lipid was not spin labeled because the protein spin labeled cysteines ites weret he focus.…”