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Biochemistry. 2014 Aug 12;53(31):5102-10. doi: 10.1021/bi500768c. Epub 2014 Jul 29.

Fluctuations of an exposed π-helix involved in lipoxygenase substrate recognition.

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Department of Biological Science, Florida State University , Tallahassee, Florida 32306-4295, United States.


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.

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