RESONANCE RAMAN-STUDY OF THE ACTIVE-SITE OF COPRINUS-CINEREUS PEROXIDASE

Resonance Raman (RR) spectra for the resting state ferric and the redu ced ferrous forms of recombinant Coprinus cinereus peroxidase (CIP), o btained 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)] a nd 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 e ach other than to HRP-C. The ferric state of CIP at neutral pH is char acteristic mainly of a five-coordinate high spin heme. However, the lo wer frequency of the v(2) mode and a higher frequency of the v(C=C) vi nyl stretching modes for. CIP as compared to CCP, indicate a higher de gree of vibrational coupling between the two modes in CIP. In addition , CIP is rather unstable under low laser power irradiation as an irrev ersible transition to a six-coordinate high spin heme followed by a se cond transition to a six-coordinate low spin heme is observed. This in stability of CIP as compared to CCP(MI) is proposed to be a consequenc e 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 l ocated above the heme Fe thereby preventing its coordination in CCP. I n 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 expen se of the band at 230 cm(-1). This shift may reflect structural change s associated with protonation of the proximal His residue. The v(Fe-Im ) stretching mode at 230 cm(-1) suggests the presence of a hydrogen-bo nded imidazole with weaker imidazolate character than in CCP(MT), wher eas the 211-cm(-1) band suggests the presence of a fairly weak hydroge n-bond interaction between the N-delta-H of the proximal His183 and th e O atom of the Asp245 side chain. The structural parameters influenci ng the frequency of the Fe-Im stretching mode are discussed.