No abstract

The second-order rate constant (k4) for the oxidation of a series of aromatic donor molecules (monosubstituted phenols and anilines) by horseradish peroxidase (HRP) compound II was examined with a stopped-flow apparatus. The electronic states of these substrates were calculated by an ab initio molecular orbital method. It was found that in both phenols and anilines log k4 values correlate well with the highest occupied molecular orbital (HOMO) energy level and the lowest unoccupied molecular orbital (LUMO) energy level, but not with the net charge or frontier electron density on atoms of these molecules. The HOMO and LUMO energy levels of phenols and anilines further showed linear relationships with Hammett's sigma values with negative slopes. Similar results were obtained in the oxidation of substrates by HRP compound I, except that the rate of reaction was much higher than in the case of HRP compound II. In addition, the rates of oxidation of phenols by compound I or II were found to be about 1000 times higher than those of anilines with similar HOMO energy levels. On the basis of these results, the mechanism of electron transfer from the substrate to the heme iron of HRP compound II is discussed.

Interaction of an iodide ion with lactoperoxidase was studied by the use of 1H NMR, 127I NMR, and optical difference spectrum techniques. 1H NMR spectra demonstrated that a major broad hyperfine-shifted signal at about 60 ppm, which is ascribed to the heme peripheral methyl protons, was shifted toward high field by adding KI, indicating the binding of iodide to the active site of the enzyme; the dissociation constant was estimated to be 38 mM at pH 6.1. The binding was further detected by 127I NMR, showing no competition with cyanide. Both 1H NMR and 127I NMR revealed that the binding of iodide to the enzyme is facilitated by the protonation of an ionizable group with a pKa value of 6.0-6.8, which is presumably the distal histidyl residue. Optical difference spectra showed that the binding of an aromatic donor molecule to the enzyme is slightly but distinctly affected by adding KI. On the basis of these results, it was suggested that an iodide ion binds to lactoperoxidase outside the heme crevice but at the position close enough to interact with the distal histidyl residue which possibly mediates electron transport in the iodide oxidation reaction.

On the basis of optical difference spectra, lactoperoxidase (LPO) was shown to form a 1:1 complex with aromatic donor molecules: resorcinol, hydroquinone, phenol, p-cresol, guaiacol, aniline, and benzohydroxamic acid. As compared with horseradish peroxidase (HRP), the values of the dissociation constant, Kd, of LPO-donor complexes were found to be 4-720-fold larger and were not greatly changed in the presence of KCN and by changes in pH in the range 4-9. The apparent enthalpy and entropy of the binding reactions were found to be -13 kJ mol-1 and -29 J mol-1 K-1, respectively, somewhat smaller (in absolute value) than the corresponding values of HRP. The difference spectra of LPO-donor complexes resembled each other, in contrast to the case of HRP, and the bindings of the donors to LPO occurred in a competitive fashion between the donors. Incubation of LPO with phenylhydrazine and hydrogen peroxide markedly depressed donor binding, the intensity of the Soret band, and the catalytic activity, probably as the result of formation of meso-phenyl derivatives of the heme. These findings suggest that the binding of aromatic donors to LPO occurs at a specific site, probably near the heme edge, where the electron transfer in the peroxidase reaction may take place.

A conspicuous adhesion of Staphylococcus aureus organisms to murine cutaneous fibroblasts and NIH/ 3T3 cells cultured in vitro and subsequent ingestion of S. aureus organisms by these fibroblasts are described.In the present experimental system, only fibroblasts-adhering S. aureus organisms were efficiently ingested by fibroblasts unlike S. epidermidis and S. saprophyticus. These findings might suggest a correlation between the pathogenesis of S. aureus and its intracellular localization in non-professional phagocytes such as fibroblasts in a special reference to its higher pathogenicity than those of coagulase negative counterparts.