Solution NMR determination of the seating(s) of meso-nitro-etioheme-1 in myoglobin: Implications for steric constraints to meso position access in heme degradation by coupled oxidation

The highly stereoselective cleavage of hemin in myoglobin by coupled oxidat ion has been attributed to steric barriers that leave more space near the a lpha- than the other meso-positions. The steric barriers near meso position s in myoglobin have been investigated by establishing the thermodynamics an d dynamics of possible seatings in the pocket of horse myoglobin of a four- fold symmetric etioheme I modified with a bulky nitro group at a single mes o position. The cyanomet complex of this reconstituted myoglobin exhibits t hree sets of H-1 NMR resonances that are linked dynamically and occur in ap proximate populations ratios of 0.82:0.10:0.08. Two dimensional H-1 NMR has been used to assign the hemin and heme pocket resonances in the major isom er in solution and to determine that the hemin is oriented with the nitro g roup at the canonical gamma -meso position of native hemin. The dominance o f this isomer is attributed to the solvent exposure of this portion of the hemin which stabilizes the highly polar nitro group. Using a combination of magnetization transfer among methyl groups of the three isomers due to "ho pping" of the hemin about it, normal, the assigned resonances of an isoelec tronic, bis-cyano complex of meso-nitro-etioheme I, and the known essential ly constant rhombic perturbation of heme pocket sites on the hyper-fine shi fts of heme methyl (Kolczak, U.; Hauksson, J. B.; Davis, N. L.; Pande, U.; de Ropp, J. S.; Langry, K. C.; Smith, K. M.; LaMar, G. N. J. Ain. Chem. Soc . 1999, 121, 835-843); the two minor isomers are shown to place their bulky nitro group at the canonical delta -meso (8%) and alpha -meso positions (1 0%). The comparable population of the isomers with nitro groups at the hydr ophobic alpha- and delta -meso positions dictates that, while the static cr ystal structure finds more room near the alpha -meso position, the deformat ion at minimal energetic expense near the alpha- and delta -meso positions is comparable. These results argue that factors other than simple steric in fluences control the selectivity of the ring cleavage in myoglobin.