A nuclear Overhauser effect study of the heme crevice in the resting state and compound I of horseradish peroxidase: evidence for cation radical delocalization to the proximal histidine.
The assignment of resolved hyperfine-shifted resonances in high-spin resting state horseradish peroxidase (HRP) and its double-oxidized reactive form, compound I (HRP-I), has been carried out by using the nuclear Overhauser effect (NOE) starting with the known heme methyl assignments in each species. In spite of the efficient spin-lattice relaxation and very broad resonances, significant NOEs were observed for all neighboring pyrrole substituents, which allowed the assignment of the elusive propionate alpha-methylene protons. In the resting state HRP, this leads directly to the identity of the proximal His-170 H beta peaks. The determination that one of the most strongly contact-shifted single proton resonances in HRP-I does not arise from the porphyrin dictates that the cation radical must be delocalized to some amino acid residue. The relaxation properties of the non-heme contact-shifted signal in HRP-I support the identity of this contributing residue as the proximal His-170. Detailed analysis of changes in both contact shift pattern and NOEs indicates that compound I formation is accompanied by a approximately 5 degree rotation of the 6-propionate group. The implication of a porphyrin cation radical delocalized over the proximal histidine for the proposed location of the solely amino acid centered radical in compound I of related cytochrome c peroxidase is discussed.
Thanabal
V
V
Department of Chemistry, University of California, Davis 95616.
La Mar
G N
GN
de Ropp
J S
JS
D002412:Cations; D006418:Heme; D006420:Hemeproteins; D006639:Histidine; D006735:Horseradish Peroxidase; D006861:Hydrogen Peroxide; D007501:Iron; D009682:Magnetic Resonance Spectroscopy; D010084:Oxidation-Reduction; D010544:Peroxidases
D002412:Cations; D006420:Hemeproteins; D006418:Heme; D006639:Histidine; D006861:Hydrogen Peroxide; D007501:Iron; D006735:Horseradish Peroxidase; D010544:Peroxidases