A nuclear Overhauser effect investigation of the molecular and electronic structure of the heme crevice in lactoperoxidase.
The proton homonuclear nuclear Overhauser effect, NOE, in conjunction with paramagnetic-induced dipolar relaxation, is utilized to assign resonances and to probe the molecular and electronic structures of the heme cavity in the low-spin cyanide complex of resting-state bovine lactoperoxidase, LPO-CN. Predominantly primary NOEs were detected in spite of the large molecular weight (approximately 78 x 10(3)) of the enzyme, which demonstrates again the advantage of paramagnetism suppressing spin diffusion in large proteins. Both of the nonlabile ring protons of a coordinated histidine are located at resonance positions consistent with a deprotonated imidazole. Several methylene proton pairs are identified, of which the most strongly hyperfine-shifted pair is assigned to the unusual chemically functionalized 8-(mercaptomethylene) group of the prosthetic group [Nichol, A. W., Angel, L. A., Moon, T., & Clezy, P. S. (1987) Biochem. J. 247, 147-150]. The large 8-(mercaptomethylene) proton contact shifts relative to that of the only resolved heme methyl signal are rationalized by the additive perturbations on the rhombic asymmetry of the functionalization of the 8-position and the alignment of the axial histidyl imidazole projection along a vector passing through pyrrole A and C of the prosthetic group. Such a stereochemistry is consistent with the resolution of only a single heme methyl group, 3-CH3, as observed.(ABSTRACT TRUNCATED AT 250 WORDS)
Thanabal
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Department of Chemistry, University of California, Davis 95616.
La Mar
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D000818:Animals; D002417:Cattle; D003486:Cyanides; D003903:Deuterium; D017666:Deuterium Oxide; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D006418:Heme; D006859:Hydrogen; D007784:Lactoperoxidase; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D010544:Peroxidases; D011487:Protein Conformation; D014867:Water
D003486:Cyanides; D014867:Water; D006418:Heme; D006859:Hydrogen; D003903:Deuterium; D007784:Lactoperoxidase; D010544:Peroxidases; D017666:Deuterium Oxide