Solution NMR characterization of an unusual distal H-bond network in the active site of the cyanide-inhibited, human heme oxygenase complex of the symmetric substrate, 2,4-dimethyldeuterohemin.

The presence of variable static hemin orientational disorder about the alpha-gamma-meso axis in the substrate complexes of mammalian heme oxygenase, together with the incomplete averaging of a second, dynamic disorder, for each hemin orientation, has led to NMR spectra with severe spectral overlap and loss of key two-dimensional correlations that seriously interfere with structural characterization in solution. We demonstrate that the symmetric substrate, 2,4-dimethyldeuterohemin, yields a single solution species for which the dynamic disorder is sufficiently rapid to allow effective and informative (1)H NMR structural characterization. A much more extensive, effective, and definitive NMR characterization of the cyanide-inhibited, symmetric heme complex of human heme oxygenase shows that the active site structure, with some minor differences, is essentially the same as that for the native protohemin in solution and crystal. A unique distal network that involves particularly strong hydrogen bonds, as well as inter-aromatic contacts, is described that is proposed to stabilize the position of the catalytically critical distal helix Asp-140 carboxylate (Liu, Y., Koenigs Lightning, L., Huang, H., Moënne-Loccoz, P., Schuller, D. J., Poulos, T. L., Loehr, T. M., and Ortiz de Montellano, P. R. (2000) J. Biol. Chem. 275, 34501-34507). The potential role of this network in placing a water molecule to stabilize the hydroperoxy species and as a template for the condensation of the distal helix upon substrate binding are discussed.