Portal de Periódicos da CAPES

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

1980; Elsevier BV; Volume: 255; Issue: 14 Linguagem: Inglês

10.1016/s0021-9258(18)43618-6

1083-351X

G. N. LA MAR, Jeffrey S. de Ropp, Kevin M. Smith, Kevin C. Langry,

Electron Spin Resonance Studies

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.Many details of the structure of the heme pocket of the resting state of horseradish peroxidase remain unresolved in spite of extensive investigations by a wide variety of physicochemical techniques (1).Unambiguously identified are the protoporphyrin prosthetic group and the trivalent oxidation state of the central iron (1).A coordinated histidyl imidazole appears reasonably well established on the basis of photooxidation (2), pH titration (3), ESR (4), and NMR (5) experiments.Although the ferric ion is generally considered to be essentially high spin, the magnetic moment of 5.23 p~ for free horseradish peroxidase (6-8) is below that expected for a pure high spin protein ( i e .5.92 p ~) .Initially, this reduced magnetic moment and the complex ESR spectra were interpreted as resulting from chemical or thermal mixtures of high and low spin forms (6-9).More recently, detailed ESR studies have led to the proposal (IO, 11) that horseradish peroxidase, at least in the neutral pH region, is a chemical mixture of high spin (hs) and a quantum-mixed spin (qrns) species, where the latter is a quantum-mechanical admixture of the S = 5/2 and