Low temperature MCD study of the species formed by photolysis of horseradish peroxidase compound I

Abstract

During the reaction of the enzyme horseradish peroxidase (HRP) with hydrogen peroxide, a highly oxidized species is formed which is known as ▪ compound I. The electronic structure of HRP compound I is considered to involve an Fe(IV) porphyrin π-cation radical [1]. Previous studies of horseradish peroxidase compound I have shown that light accelerates the spontaneous conversion of compound I to compound II at room temperature, yielding, finally, the native enzyme [2]. However, photolysis at low temperatures ( i.e. those less than the glassing temperature of the solvent) produces a photochemical product with optical properties which are close to, but not identical to, those of compound II [2–4]. Although, magnetic circular dichroism (MCD) spectroscopy is closely related to optical absorption spectroscopy, the data obtained provide considerably more information about the ground and excited state electronic configurations than is obtained from the absorption spectrum alone. In this paper we describe MCD data recorded between 4 K and 50 K following the photolysis of HRP compound I at 80 K. Figure 1 shows the 4 K and K MCD spectra of the HRP compound I photochemical product which was prepared by an exhaustive photolysis of compound I at 80 K. The 50 K spectrum is very similar to the MCD spectra of HRP compound II which have been previously reported for 127 K and 208 K [5]. Both sets of spectra in the visible region contain features which are quite unlike those of a ferric heme with either a S = 5 2 or 1 2 spin state. However, like the MCD spectra of ferric hemes, the spectrum of the photochemical species is very temperature dependent and is dominated Faraday C terms. Fig. 2 shows the temperature dependence of the peak-to-trough intensity for the major features in the B or Soret regions and the Q or α band regions of the MCD spectrum of the heme in the photochemical product. The almost linear ▪ relationship between the intensity and the inverse of the absolute temperature suggest that the ground state in the iron porphyrin species which is formed photochemically is obtained degenerate. The non-linearity of this relationship at very low temperatures and the high magnetic field (4.58 T) used in this study most likely arises from simple Boltzmann saturation effects. Various structures have been suggested for the product of the photolysis of HRP compound I [2, 4, 6, 7,]. The close similarity between the optical absorption and MCD spectra of the photolysis product and HRP compound II suggests that the heme retains the S = 1 Fe(IV) porphyrin electronic structure following photolysis and that the porphyrin is reduced from the 17 π-electron cation radical of compound I to the stable 18 π electron configuration as in compound II [8, 9]. In addition, the strong temperature dependence of the MCD intensity observed in the spectra of the photochemical product indicates that significant coupling takes place between the paramagnetic iron and the diamagnetic porphyrin π system.