Determination by 1H NMR of the Orientation of Modified Hemes Incorporated into Horseradish Peroxidase. Evidence for Steric Clamping of a Vinyl Group by the Protein

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Proton NMR spectroscopy of horseradish peroxidase has been utilized to probe the interaction between the heme side chains of pyrroles I and II and the apo protein. Reconstitution of apo protein with both equivalent 2,4-disubstituted and nonequivalent 2,4-substituted deuterohemins provides a sensitive probe of the heme-protein interactions of pyrroles I and II. Use of specifically deuterated 2,4-modified hemins permits the assignment of heme peaks, which in turn determines the orientation of the heme in the heme pocket. It was determined that 2,4-diacetyldeuterohemin-, 4-acetyldeuterohemin- and 4-vinyldeuterohemin-reconstituted horseradish peroxidase all exist with the heme orientation adopted by native protohemin, while deuterohemin-, 2-acetyldeuterohemin-, and 2-vinyldeuterohemin-reconstituted horseradish peroxidase exist with the heme predominantly in an orientation that is rotated 180° about the heme α-γ axis. From this it was concluded that the heme pocket of the native protein contains more room for the 2,4-vinyl side chains than the 1,3-methyl groups. Furthermore, the preference for the 4-acetyl- and 4-vinyldeuterohemins to exist with the heme in the native orientation, while the 2-acetyl- and 2-vinyldeuterohemins exist predominantly in the 180° flipped form, argues that the 2-vinyl side chain is more sterically constrained than the 4-vinyl side chain, results that are supported by previous investigations of the 2-α-vinyl resonance in HRP compound I. A possible role of the steric constraint of the vinyl groups in stabilizing HRP compound I is discussed. © 1983, American Chemical Society. All rights reserved.

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Journal of the American Chemical Society

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