<sup>1</sup>H NMR Study of the Influence of Hemin Vinyl→Methyl Substitution on the Interaction between the <i>C</i>-Terminus and Substrate and the “Aging” of the Heme Oxygenase from <i>Neisseria meningitidis</i>:  Induction of Active Site Structural Heterogeneity by a Two-Fold Symmetric Hemin<sup>†</sup>

Solution <sup>1</sup>H NMR has been used to characterize the active site molecular and electronic structure of the cyanide-inhibited 2,4-dimethyldeuterohemin complex of the heme oxygenase from <i>Neisseria meningitidis</i> (<i>Nm</i>HO) with respect to the mode of interaction of the <i>C</i>-terminus with the substrate and the spontaneous “aging” of <i>Nm</i>HO that results in the cleavage of the <i>C</i>-terminal Arg208-His209 dipeptide. The structure of the portion involving residues Ala12-Phe192 is found to be essentially identical to that of the protohemin complex in either solution or crystal. However, His207 from the <i>C</i>-terminus is found to interact strongly with the substrate 1CH<sub>3</sub>, as opposed to the 8CH<sub>3</sub> in the protohemin complex. The different mode of interaction of His207 with the alternate substrates is attributed to the 2-vinyl group of protohemin sterically interfering with the optimal orientation of the proximal helix Asp27 carboxylate that serves as acceptor to the strong H-bond by the peptide of His207. The 2,4-dimethyldeuterohemin HO complex “ages” in manner similary to that of protohemin, (Liu, Y., Ma, L.-H., Satterlee, J.D., Zhang, X., Yoshida, T., and La Mar, G. N., (2006) <i>Biochemistry</i> <i>45</i>, 3875−3886) with mass spectrometry and <i>N</i>-terminal sequencing indicating that the Arg208-His209 dipeptide is cleaved. The 2,4-dimethyldeuterohemin complex of WT HO populates an equilibrium isomer stabilized in low phosphate concentration for which the axial His imidazole ring is rotated by ∼20° from that in the WT. The His ring reorientation is attributed to Asp24 serving as the H-bond acceptor to the His207 peptide NH, rather than to the His23 ring N<sub>δ</sub>H as in the crystals. The functional implications of the altered <i>C</i>-terminal interaction with substrate modification are discussed.