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

Abstract

The addition of exogenous ligands to the ferric and ferrous states of yeast cytochrome c peroxidase (CCP) is investigated with magnetic circular dichroism (MCD) at 4°C to determine the effect the protein environment may exercise on spectral properties. The MCD spectrum of each derivative is directly compared to those of analogous forms of horseradish peroxidase (HRP) and myoglobin (Mb), two well-characterized histidine-ligated heme proteins. The ferric azide adduct of CCP is a hexacoordinate, largely low-spin species with an MCD spectrum very similar to that of ferric azide HRP. This complex displays an MCD spectrum dissimilar from that of the Mb derivative, possibly because of the stabilizing interaction between the azide ligand and the distal arginine of CCP (Arg 48). For the ferric fluoride derivative all three proteins display varied MCD data, indicating that the differences in the distal pocket of each protein influences their respective MCD characteristics. The MCD data for the cyanoferric complexes are similar for all three proteins, demonstrating that a strong field ligand bound in the sixth axial position dominates the MCD characteristics of the derivative. Similarly, 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 and cyanide complexes of ferrous CCP gives derivatives with MCD spectra similar 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 MCD spectral similarity to that of ferrous-CO HRP but not Mb, indicating that interactions between the ligand and the distal residues affects the MCD characteristics. Examination of alkaline (pH 9.7) deoxyferrous CCP indicates that a pH dependent conformational change has occurred, leading to a coordination structure similar to that of ferrous cytochrome b₅, a known bis-histidine complex. Exposure of this complex to CO further confirms that a conformational change has taken place in that the MCD spectral characteristics of the resulting complex are similar to those of ferrous-CO Mb but not ferrous-CO HRP. © 1999 John Wiley & Sons, Inc. Biospectroscopy 5: S42–S52, 1999