Alycen E. Pond

Spectroscopic study of the compound ES and the oxoferryl compound II states of cytochrome c peroxidase: comparison with the compound II of horseradish peroxidase☆

Magnetic circular dichroism (MCD) spectroscopic data at −30°C are reported for the oxoferryl compound II state of cytochrome c peroxidase (CcP), which was generated by hydrogen peroxide oxidation of ferrous CcP, in comparison with compound ES (an oxoferryl heme with a protein based-radical) of CcP and compound II (an oxoferryl heme) of horseradish peroxidase (HRP-II). Detailed spectral analyses reveal that the protein-based free radical found in CcP-ES does not significantly perturb the electronic environment of the heme as measured by electronic absorption and MCD spectroscopy. Thus, the spectra of the compounds I and II (oxoferryl) states of CcP are similar. The compound I state of HRP (HRP-1), on the other hand, contains an oxoferryl heme coupled to a porphyrin π-cation radical. As a consequence, HRP-I and HRP-II are well known to have quite distinctive spectra. The similarity of the spectral properties of the two active species of CcP, and the contrasting dissimilarity between the spectral properties of the two active high valent species of HRP, clearly indicates the importance of the protein structure surrounding the heme group in determining the spectral properties of the active species.

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 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.

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 dichroism (MCD) at 4 degrees 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.

Initial characterization of the ferric H175G cytochrome c peroxidase cavity mutant using magnetic circular dichroism spectroscopy: phosphate from the buffer as an axial ligand

Abstract Electronic absorption and magnetic circular dichroism (MCD) data are reported for the H175G cavity mutant of yeast cytochrome c peroxidase (H175G CCP) in its ligand-free ferric state at 4 °C under slightly acidic conditions (pH 5.9). Initial analysis of the MCD spectrum of this state suggested a five-coordinate structure with ligation by an aspartate or glutamate. However, examination of the crystal structure of wild-type CCP revealed no plausible carboxylate-containing amino-acid residues close enough to the heme iron to serve as the ligand. An alternative interpretation of the MCD data is that the phosphate ion from the buffer is bound to the ferric heme iron. A phosphate group is similar in electronic character to a carboxylate and could give an MCD signal similar to that of a carboxylate-bound heme. Phosphate coordination to the ferric heme iron, albeit trans to imidazole, has recently been seen in a 2.0-A-resolution crystal structure of imidazole-bound ferric H175G CCP in phosphate buffer [Biochemistry 40 (5) (2001) 1265]. To investigate the validity of phosphate ligation, the pH 5.9 species in the 100 mM phosphate buffer was exchanged into the pH 5.9, 100 mM MES (2-[N-morpholino]ethanesulfonic acid) buffer. The MCD spectrum of the species in the MES buffer was spectrally distinct from that in the phosphate buffer, indicating that the original spectrum depends on the presence of a phosphate ion. We conclude that in the phosphate buffer, the exogenous ligand-free ferric state of the H175G CCP cavity mutant is actually coordinated by a phosphate ion from the buffer.