Influence of the distal his in imparting imidazolate character to the proximal his in heme peroxidase: (1)h NMR spectroscopic study of cyanide-inhibited his42-->ala horseradish peroxidase.
The functional higher oxidation states of heme peroxidases have been proposed to be stabilized by the significant imidazolate character of the proximal His. This is induced by a "push-pull" combination effect produced by the proximal Asp that abstracts ("pulls") the axial His ring N(delta)H, along with the distal protonated His that contributes ("pushes") a strong hydrogen bond to the distal ligand. The molecular and electronic structure of the distal His mutant of cyanide-inhibited horseradish peroxidase, H42A-HRPCN, has been investigated by NMR. This complex is a valid model for the active site hydrogen-bonding network of HRP compound II. The (1)H and (15)N NMR spectral parameters characterize the relative roles of the distal His42 and proximal Asp247 in imparting imidazolate character to the axial His. 1D/2D spectra reveal a heme pocket molecular structure that is highly conserved in the mutant, except for residues in the immediate proximity of the mutation. This conserved structure, together with the observed dipolar shifts of numerous active site residue protons, allowed a quantitative determination of the orientation and anisotropies of the paramagnetic susceptibility tensor, both of which are only minimally perturbed relative to wild-type HRPCN. The quantitated dipolar shifts allowed the factoring of the hyperfine shifts to reveal that the significant changes in hyperfine shifts for the axial His and ligated (15)N-cyanide result primarily from changes in contact shifts that reflect an approximately one-third reduction in the axial His imidazolate character upon abolishing the distal hydrogen-bond to the ligated cyanide. Significant changes in side chain orientation were found for the distal Arg38, whose terminus reorients to partially fill the void left by the substituted His42 side chain. It is concluded that 1D/2D NMR can quantitate both molecular and electronic structural changes in cyanide-inhibited heme peroxidase and that, while both residues contribute, the proximal Asp247 is more important than the distal His42 in imparting imidazole character to the axial His 170.
de Ropp
Jeffrey S
JS
Contribution from the Department of Chemistry and NMR Facility, University of California, Davis, California 95616, USA.
Sham
Simon
S
Asokan
Anbanandan
A
Newmyer
Sherri
S
Ortiz de Montellano
Paul R
PR
La Mar
Gerd N
GN
D000409:Alanine / Q000737:chemistry; D003486:Cyanides / Q000737:chemistry* / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D006418:Heme / Q000737:chemistry* / Q000378:metabolism; D006639:Histidine / Q000737:chemistry*; D006735:Horseradish Peroxidase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D006860:Hydrogen Bonding; D007093:Imidazoles / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular; D010544:Peroxidases / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation
D003486:Cyanides; D004791:Enzyme Inhibitors; D007093:Imidazoles; D006418:Heme; D006639:Histidine; D006735:Horseradish Peroxidase; D010544:Peroxidases; D000409:Alanine