Substituted Hemins as Probes for Structure-Function Relationships in Horseradish Peroxidase
DiNello, R.K. and David DolphinH.
J. Biol. Chem.
Low-temperature visible spectra of compounds I from peroxidases reconstituted with protohemin, 2-formyl-4-vinyldeuterohemin, 2-vinyl-4-formyldeuterohemin, 2,4-dimethyldeuterohemin, and 2,4-diacetyldeuterohemin reveal that these Fe(IV) porphyrin π-cation radicals take the 2A2u or peroxidase-type electronic ground state. Compound I of deuterohemin horseradish peroxidase, however, takes the 2A1u or catalase type π-cation radical electronic ground state. Since deuterohemin horseradish peroxidase possesses no catalase activity, the structure of the peroxidase apoprotein (other than those factors which might influence the compound I π-cation radical ground state) is concluded to play the major role in determining the reactivity of compound I toward H-donors. Studies on peroxidases substituted with the hemins 2-formyl-4-vinyldeuterohemin, 2-vinyl-4-formyldeuterohemin, 2,4-dimethyldeuterohemin, and mesohemin reveal that isoelectonic hemins differentially interact with the peroxidase apoprotein. The hemin 2- and 4-substituents are therefore concluded to interact sterically with the horseradish peroxidase apoprotein. Whereas a variety of 2- and 4-substituted hemins were observed to bind rapidly with apo-horseradish peroxidase to form active substituted enzymes, small changes in the substituents in the 6- and 7-positions had drastic effects on the rates of binding of apoperoxidase and the activities of the reconstituted enzymes. Even addition of a single methylene to form butyrate instead of propionate side chains drastically altered the rate of binding of the hemin and the activity of the substituted enzyme. It therefore appears that whereas the 2-, 4-, 6-, and 7-substituents of the hemins in horseradish peroxidase all interact with the protein, the polypeptide chain possesses more conformational flexibility in the area which binds the 2- and 4-substituents.