title: Implication for using heme methyl hyperfine shifts as indicators of heme seating as related to stereoselectivity in the catabolism of heme by heme oxygenase: in-plane heme versus axial his rotation.
abstract: The triple mutant of the solubilized, 265-residue construct of human heme oxygenase, K18E/E29K/R183E-hHO, has been shown to redirect the exclusive alpha-regioselectivity of wild-type hHO to primarily beta,delta-selectivity in the cleavage of heme (Wang, J., Evans, J. P., Ogura, H., La Mar, G. N., and Ortiz de Montellano, P. R. (2006) Biochemistry 45, 61-73). The 1H NMR hyperfine shift pattern for the substrate and axial His CbetaH's and the substrate-protein contacts of the cyanide-inhibited protohemin and 2,4-dimethyldeuterohemin complexes of the triple mutant have been analyzed in detail and compared to data for the WT complex. It is shown that protein contacts for the major solution isomers for both substrates in the mutant dictate approximately 90 degrees in-plane clockwise rotation relative to that in the WT. The conventional interpretation of the pattern of substrate methyl hyperfine shifts, however, indicates substrate rotations of only approximately 50 degrees . This paradox is resolved by demonstrating that the axial His25 imidazole ring also rotates counterclockwise with respect to the protein matrix in the mutant relative to that in the WT. The axial His25 CbetaH hyperfine shifts are shown to serve as independent probes of the imidazole plane orientation relative to the protein matrix. The analysis indicates that the pattern of heme methyl hyperfine shifts cannot be used alone to determine the in-plane orientation of the substrate as it relates to the stereospecificity of heme cleavage, without explicit consideration of the orientation of the axial His imidazole plane relative to the protein matrix.
authors: Department of Chemistry, University of California, Davis, California 95616, USA.
authors: Hiroshi
authors: H
authors: Ogura
authors: John P
authors: JP
authors: Evans
authors: Paul R Ortiz
authors: PR
authors: de Montellano
authors: Gerd N
authors: GN
authors: La Mar
mesh_terms: D001664:Biliverdine / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D006418:Heme / Q000031:analogs & derivatives / Q000737:chemistry* / Q000378:metabolism*; D006419:Heme Oxygenase (Decyclizing) / Q000235:genetics / Q000378:metabolism*; D006427:Hemin / Q000737:chemistry; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: C099547:verdoheme; D006418:Heme; D006427:Hemin; D006419:Heme Oxygenase (Decyclizing); D001664:Biliverdine