Abstract
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.
Highlights
Changeable proton in horseradish peroxidase but not Severarel cent spectroscopic studies, argue in metmyoglobins is suggested to arise from strong stronglyagainsta coordinatedwater, since the bulk water hydrogen bonding between the coordinated imidazole protons experience minimal enhanced relaxation (20) and “0 and some unspecified protein acceptor residue in the hyperfine splitting was found to be absent in the ESR spectra former protein
Unambiguouslyidentified are the Nuclear magnetic resonance is in many ways protoporphyrin prosthetic group and the trivalent oxidation well suited for elucidating the detailed structureof the active state of the central iron (1).A coordinated histidyl imidazole site of paramagnetic hemoproteinsbecause of the strong couappears reasonably well established on thebasis of photooxi- pling between the iron unpaired spins and the nuclei of the dation (2), pH titration (3), ESR (4), and NMR (5) experi- appended ligands such as the porphyrin, proximal histidine, ments
The protein was purified by column horseradish peroxidase which contain the resolved hyperfine shifted resonances below 12 and above 0 ppm from DSS are illustrated in Fig. 2 a t several temperatures
Summary
The protein was purified by column horseradish peroxidase which contain the resolved hyperfine shifted resonances below 12 and above 0 ppm from DSS are illustrated in Fig. 2 a t several temperatures. These peaks are generally found in the region 30 to 60 ppm from DSS in all ferric models (27-29), so t h a t of the six remaining resolved downfield single proton resonances,h tom, four are very likely 6,7-H,,’s.
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