Temperature dependence in the MCD spectrum of horseradish peroxidase compound I

Abstract

Ater considerable experimental study, the analysis of ENDOR [1, 2], and magnetic circular dichroism (MCD) spectra of horseradish peroxidase (HRP) compound I [3], as well as the MCD spectra of a series of porphyrin π-cation radical species [4] has led to the confirmation that the electronic configuration of the heme in the compound I species was that of an Fe(IV) porphyrin π-cation complex. While the MCD data of the enzyme were measured at 273 K, and these spectra can be obtained at low temperatures, the ENDOR [1] and EPR [5] signals can only be detected below 30 K. The MCD experiment is very sensitive to ground state degeneracy and in this paper spectra of HRP compound I are discussed that were obtained between liquid helium temperatures and 100 K. Figure 1 shows how the reduction in temperature has a far more pronounced effect on the MCD spectrum of HRP compound I than on the comparable absorption spectrum. While there are only slight changes in the MCD spectrum between 273 K and ▪ 30 K, there are very dramatic changes between 30 K and 4 K. Below 30 K, there is a loss in intensity at 660 nm with a corresponding increase in the 640 nm band and an increase in the dominance of the 300 nm to 500 nm region by the 420 nm and 460 nm bands. The temperature dependent band at 420 nm appears to be derived from a small impurity of the photochemical product of HRP compound I [6] which is formed during preparation of the sample and as a consequence of the visible and UV light used to measure the MCD spectra. The most striking feature of the spectrum below 30 K is the is the apparent relationship between the 640 nm and 660 nm bands, and the simple intensity increase with inverse temperature that is observed in the 460 nm and 640 nm bands. The latter effect is characteristic of a C term and thus indicates the presence of an orbitally degenerate ground state. Coupling between the S = 1 iron and the S = 1 2 porphyrin to form the degenerate ground state as a set of three Kramers doublets has been used to explain the observations of the temperature dependence of the EPR spectrum [5]; this hypothesis can also be used to describe the appearance of the MCD C term [7]. The lack of temperature dependence above 30 K suggests that the iron and the porphyrin radical are not now strongly coupled together. These data also suggest that a structural change has occured at the low temperatures which results in the temperature dependence in the MCD spectrum as the result of a change in the coupling between the S = 1 iron and the S = 1 2 π-cation porphyrin radical, and the formation of a degenerate ground state.