Proton NMR Investigation of Substrate-Bound Heme Oxygenase: Evidence for Electronic and Steric Contributions to Stereoselective Heme Cleavage

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The substrate-bound form of the enzyme heme oxygenase (HO), which catalyzes the stereospecific α-meso bridge cleavage of hemin to yield biliverdin IXα, has been investigated by 1H NMR in both its primarily high-spin and its cyanide-inhibited low-spin forms. Both derivatives yield 1H NMR spectra indicative of extensive heterogeneity that is largely resolved when a 2-fold-symmetric hemin substrate is bound. The structural origin of the heterogeneity is shown to result from ∼ 1:1 isomeric binding of the native hemin substrate in the binding pocket. The substrate orientational disorder is about the α,γ-meso axis, as established on the basis of 2D NMR experiments that identify characteristic aromatic van der Waals contact in the substrate binding pocket. The isomeric substrate-HO complexes exhibit differential cyanide affinity, and the ratio of isomers is sensitive to the hemin 2,4-substituents. The assignment of hemin signals by isotopic labeling and 2D NMR methods reveals a contact shift pattern that reflects an unusual hemin electronic structure that is characterized by large differences in delocalized spin density for the two positions within a given pyrrole, rather than the more conventional large differences between adjacent pyrroles. This pattern of spin density delocalized primarily to the pyrrole positions adjacent to the α,γ-meso axis can be rationalized by postulating a direct electronic perturbation of the hemin by the protein matrix in the form of an anionic side chain close to the α-meso carbon. Similar influences on hemin electronic structure, in the form of chemical substitution of the meso positions, have been observed in iron porphyrin compounds and successfully modeled by simple molecular orbital theory (Tan et al., 1994). This is interpreted as evidence for a direct electronic effect by HO to activate the α-meso position for electrophilic rather than nucleophilic attack. The unique contact shift pattern is present to different degrees for the two hemin orientations, is strongly pH dependent, and is largely abolished at acidic pH. Portions of several heme pocket residues are located, and it is shown that the pattern of the dipolar shifts for these residues, which likely reflects the distal steric influence on the tilt of the coordinated cyanide, differs significantly for the two substrate orientations. The variable electronic and steric influences of the protein matrix on the two hemin substrate orientations suggest heterogeneous, pH-dependent kinetics, but unaltered stereoselectivity in the enzyme reaction, and may account for the wide range of activity of HO with respect to a variety of peripherally modified hemins. © 1994, American Chemical Society. All rights reserved.

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