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)