Title
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
Document Type
Article
Publication Date
1-1-1983
Abstract
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.
Publication Source (Journal or Book title)
Journal of the American Chemical Society
First Page
4576
Last Page
4580
Recommended Citation
La Mar, G., de Ropp, J., Smith, K., & Langry, K. (1983). 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. Journal of the American Chemical Society, 105 (14), 4576-4580. https://doi.org/10.1021/ja00352a012