Proton nuclear magnetic resonance study of the electronic and molecular structure of the heme crevice in horseradish peroxidase.

High field proton nuclear magnetic resonance spectroscopy was used to investigate the electronic and molecular structure of the ferric heme in the resting state of horseradish peroxidase. Deuterium labeling of selected positions of hemin and deuterohemin which were subsequently reconstituted into apo-horseradish peroxidase yielded hyperfine shift patterns for the prosthetic group which are consistent with a ferric porphyrin exhibiting appreciable S = 3/2 character in a quantum mixed spin state. All resolved resonances with significant hyperfine shifts can be accounted for by the porphyrin and a proximal histidyl imidazole, although a sixth ligand from the protein cannot be definitely eliminated. The extremely slow exchange rate with bulk water of the proximal histidyl imidazole exchangeable proton and the absence of deviations from Curie behavior for the porphyrin vinyl and propionic acid proton hyperfine shifts indicate a buried heme crevice which is more rigid than in metmyoglobin. The observation of significant deviations from Curie behavior of the proximal histidyl imidazole exchangeable proton in horseradish peroxidase but not in metmyoglobins is suggested to arise from strong hydrogen bonding between the coordinated imidazole and some unspecified protein acceptor residue in the former protein.