Resonance Raman Study of the Active Site of Coprinus cinereus Peroxidase

Resonance Raman (RR) spectra for the resting state ferric and the reduced ferrous forms of recombinant Coprinus cinereus peroxidase (CIP), obtained with different excitation wavelengths and in polarized light, are reported. The spectra are compared with those obtained previously for cytochrome c peroxidase expressed in Escherichia coli [(CCP(MI)] and horseradish peroxidase (HRP-C). Although the enzymic properties of CIP and HRP-C are similar, the RR data show that, in terms of the heme cavity structures, CIP and CCP(MI) are much more closely related to each other than to HRP-C. The ferric state of CIP at neutral pH is characteristic mainly of a five-coordinate high spin heme. However, the lower frequency of the v(2) mode and a higher frequency of the v(C=C) vinyl stretching modes for. CIP as compared to CCP, indicate a higher degree of vibrational coupling between the two modes in CIP. In addition, CIP is rather unstable under low laser power irradiation as an irreversible transition to a six-coordinate high spin heme followed by a second transition to a six-coordinate low spin heme is observed. This instability of CIP as compared to CCP(MI) is proposed to be a consequence of the presence of a distal Phe54 in CIP rather than the homologous Trp51 in CCP, as Trp51 is hydrogen-bonded to a distal water molecule located above the heme Fe thereby preventing its coordination in CCP. In CIP the Fe-II-His RR band has two components with frequencies at 230 and 211 cm(-1). The 230-cm(-1) band is the more intense at neutral pH, whereas at alkaline pH the band at 211 cm(-1) increases at the expense of the band at 230 cm(-1). This shift may reflect structural changes associated with protonation of the proximal His residue. The v(Fe-Im) stretching mode at 230 cm(-1) suggests the presence of a hydrogen-bonded imidazole with weaker imidazolate character than in CCP(MT), whereas the 211-cm(-1) band suggests the presence of a fairly weak hydrogen-bond interaction between the N-delta-H of the proximal His183 and the O atom of the Asp245 side chain. The structural parameters influencing the frequency of the Fe-Im stretching mode are discussed.