title: 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.
abstract: Electronic absorption and magnetic circular dichroism (MCD) spectroscopic data at 4 degrees C are reported for exogenous ligand-free ferric forms of cytochrome c peroxidase (CCP) in comparison with two other histidine-ligated heme proteins, horseradish peroxidase (HRP) and myoglobin (Mb). In particular, 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 YCCP, and alkaline CCP (MKT). The present study demonstrates that, while having similar electronic absorption spectra, 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/water hexa-coordinate). The electronic absorption spectrum of ferric CCP (MKT) is similar to those of the penta-coordinate states of ferric YCCP and ferric HRP including a CT band at 645 nm. However, its MCD spectrum shows a small trough at 583 nm that is absent in the analogous spectra of YCCP and HRP. Instead, this trough is similar to that seen for ferric myoglobin at about 585 nm, and is attributed (following spectral simulations) to a minor contribution (< or = 5%) in the spectrum of CCP (MKT) from a hexa-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-spin species including both His/hydroxide and bis-His species. The crystal structure of a spectroscopically similar sample of CCP (MKT) (lambda CT = 637 nm) solved at 2.0 A resolution is consistent with His/hydroxide coordination. Alkaline CCP (pH 9.7) is proposed to exist as a mixture of hexa-coordinate, predominantly low-spin complexes with distal His 52 and hydroxide acting as distal ligands based on MCD spectral comparisons.
authors.affiliation: Department of Chemistry and Biochemistry, University of South Carolina, Columbia 29208, USA.
authors.forename: A E
authors.initials: AE
authors.lastname: Pond
authors.forename: M
authors.initials: M
authors.lastname: Sono
authors.forename: E A
authors.initials: EA
authors.lastname: Elenkova
authors.forename: D E
authors.initials: DE
authors.lastname: McRee
authors.forename: D B
authors.initials: DB
authors.lastname: Goodin
authors.forename: A M
authors.initials: AM
authors.lastname: English
authors.forename: J H
authors.initials: JH
authors.lastname: Dawson
mesh_terms: D000818:Animals; D020134:Catalytic Domain; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D003578:Cytochrome-c Peroxidase / Q000737:chemistry*; D006418:Heme / Q000737:chemistry; D006735:Horseradish Peroxidase / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D009211:Myoglobin / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013053:Spectrophotometry
chemical_list: D008024:Ligands; D009211:Myoglobin; D011994:Recombinant Proteins; D006418:Heme; D006735:Horseradish Peroxidase; D003578:Cytochrome-c Peroxidase