Magnetic circular dichroism studies of the active site heme coordination sphere of exogenous ligand-free ferric cytochrome c peroxidase from yeast: effects of sample history and pH

Electronic absorption and magnetic circular dichroism (MCD) spectroscopic d ata at 4 degrees C are reported for exogenous ligand-free ferric forms of c ytochrome c peroxidase (CCP) in comparison with two other histidine-ligated heme proteins, horseradish peroxidase (HRP) and myoglobin (Mb). In particu lar, we have examined the ferric states of yeast wild-type CCP (YCCP), CCP( MKT) which is the form of the enzyme that is expressed in and purified from E. coli, and contains Met-Lys-Thr (MKT) at the N-terminus, CCP(MKT) in the presence of 60% glycerol, lyophilized YCCPI and alkaline CCP(MKT). The pre sent study demonstrates that, while having similar electronic absorption sp ectra, the MCD spectra of ligand-free ferric YCCP and CCP(MKT) are somewhat varied from one another. Detailed spectral analyses reveal that the ferric form of YCCP, characterized by a long wavelength charge transfer (CT) band at 645 nm, exists in a predominantly penta-coordinate state with spectral features similar to those of native ferric HRP rather than ferric Mb (His/w ater hexa-coordinate). The electronic absorption spectrum of ferric CCP(MKT ) is similar to those of the penta-coordinate states of ferric YCCP and fer ric HRP including a CT band at 645 nm. However, its MCD spectrum shows a sm all trough at 583 nm that is absent in the analogous spectra of YCCP and HR P. Instead, this trough is similar to that seen for ferric myoglobin at abo ut 585 nm, and is attributed (following spectral simulations) to a minor co ntribution (less than or equal to 5%) in the spectrum of CCP(MKT) from a he xa-coordinate low-spin species in the form of a hydroxide-ligated heme. The MCD data indicate that the lyophilized sample of ferric YCCP (lambda(CT) = 637 nm) contains considerably increased amounts of hexa-coordinate low-spi n species including both His/hydroxide and bis-His species. The crystal str ucture of a spectroscopically similar sample of CCP( MKT) (lambda(CT) = 637 nm) solved at 2.0 Angstrom resolution is consistent with His/hydroxide coo rdination. Alkaline CCP (pH 9.7) is proposed to exist as a mixture of hexa- coordinate, predominantly low-spin complexes with distal His 52 and hydroxi de acting as distal ligands based on MCD spectral comparisons. (C) 1999 Els evier Science Inc. All rights reserved.