Circular dichroism studies on native and artificial horseradish peroxidases.

The circular dichroism spectra of native and recombined vinyl-(protojhematin horseradish peroxidase are identical, which confirms the proper position of the reintroduced group. The same observation and conclusion applies to the light absorption spectra. Circular dichroism and light absorption spectra of seven artificial peroxidases, prepared from apoperoxidase and 2,4substituted hematins, have been studied. The Z&substituents are vinyl, 1-propenyl, I-butenyl, ethyl, hydroxyethpl and acetyl groups, and hydrogen. The elongation of the vinyl groups to I-propenyl and 1-butenyl in the hematin does not prevent the proper orientation of the prosthetic group in the holoenzymes. In the Soret region, the vinyl-(proto)-, propenyland butenylhematin peroxidases give intense, positive Cotton effects which can be resolved in two ways. One procedure yields two strong, positive Gaussian curves at a constant spacing and two smaller Gaussian curves. The other procedure yields one huge, positive Gaussian curve and two minor, negative bands, one of which occurs at the same position as the absorbance maximum. The second procedure is preferred since it requires a lower total number of Gaussian curves and can be applied to several artificial peroxidases. The intense, positive Cotton effect in the Soret region as well as a strong, negative ellipticity at 280 to 290 nm are unique to the alkenylhematin peroxidases. This suggests that the side chain double bonds and aromatic amino acid residues interact in a specific way. The possible origins of some transitions are discussed. Saturation of the Z&side chains drastically changes the circular dichroism spectra in the near ultraviolet and violet light, indicating that the interaction of the hematin with the protein differs from that in the alkenylhematin peroxidases. The steric effects of the side chains are discussed. The alkenylhematin peroxidases, which show the unique, positive Cotton effect in the Soret region, give a higher rate of formation of Compound I with hydrogen peroxide than