Influence of protein environment on magnetic circular dichroism spectral properties of ferric and ferrous ligand complexes of yeast cytochrome c peroxidase

The addition of exogenous ligands to the ferric and ferrous states of yeast cytochrome c peroxidase (CCP) is investigated with magnetic circular dichr oism (MCD) at 4 degrees C to determine the effect the protein environment m ay exercise on spectral properties. The MCD spectrum of each derivative is directly compared to those of analogous forms of horseradish peroxidase (HR P) and myoglobin (Mb), two well-characterized histidine-ligated heme protei ns. The ferric azide adduct of CCP is a hexacoordinate, largely low-spin sp ecies with an MCD spectrum very similar to that of ferric azide HRP. This c omplex displays an MCD spectrum dissimilar from that of the Mb derivative, possibly because of the stabilizing interaction between the azide ligand an d the distal arginine of CCP (Arg 48). For the ferric fluoride derivative a ll three proteins display varied MCD data, indicating that the differences in the distal pocket of each protein influences their respective MCD charac teristics. The MCD data for the cyanoferric complexes are similar for all t hree proteins, demonstrating that a strong field ligand bound in the sixth axial position dominates the MCD characteristics of the derivative. Similar ly, the ferric NO complexes of the three proteins show MCD spectra similar in feature position and shape, but vary somewhat in intensity. Reduction of CCP at neutral pH yields a typical pentacoordinate high-spin complex with an MCD spectrum similar to that of deoxyferrous HRP. Formation of the NO an d cyanide complexes of ferrous CCP gives derivatives with MCD spectra simil ar to the analogous forms of HRP and Mb in both feature position and shape. Addition of CO to deoxyferrous CCP results in a ferrous-CO complex with MC D spectral similarity to that of ferrous-CO HRP but not Mb, indicating that interactions between the ligand and the distal residues affects the MCD ch aracteristics. Examination of alkaline (pH 9.7) deoxyferrous CCP indicates that;a pH dependent conformational change has occurred, leading to a coordi nation structure similar to that of ferrous cytochrome b(5), a known bis-hi stidine complex. Exposure of this complex to CO further confirms that a con formational change has taken place in that the MCD spectral characteristics of the resulting complex are similar to those of ferrous-CO Mb but not fer rous-CO HRP. (C) 1999 John Wiley & Sons, Inc.