Circular dichroism studies on cytochrome peroxidase from baker's yeast (Saccharomyces cerevisiae)

Sievers, Gunnel

doi:10.1016/0005-2795(78)90067-3

Cited by 23 publications

(10 citation statements)

References 50 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The high spin to low spin transition can also be detected from the CD spectra of native CCP(II) in the near-UV wavelength region (240-380 nm, Figure 5B). The optical activity of heme proteins in this region is believed to arise primarily from the heme group and the aromatic side chains (Chen et al, 1974;Sievers, 1978;Myer, 1985). At pH = 7.30, a prominent positive maximum position is seen at 262 nm associated with a huge negative ellipticity at 300 nm and a shoulder at 288 nm, which at very close to the previous published results (Sievers, 1978).…”

Section: T H I S C O N T E N T Isupporting

confidence: 88%

“…The optical activity of heme proteins in this region is believed to arise primarily from the heme group and the aromatic side chains (Chen et al, 1974;Sievers, 1978;Myer, 1985). At pH = 7.30, a prominent positive maximum position is seen at 262 nm associated with a huge negative ellipticity at 300 nm and a shoulder at 288 nm, which at very close to the previous published results (Sievers, 1978). Similar spectra were obtained at pH = 8.50 and pH = 9.70.…”

Section: T H I S C O N T E N T Isupporting

confidence: 88%

See 1 more Smart Citation

Formation and photolability of low-spin ferrous cytochrome c peroxidase at alkaline pH

Wang¹,

Boldt²,

Ondrias³

1992

Biochemistry

View full text Add to dashboard Cite

The ferrous form of native cytochrome c peroxidase (CCP) is known to undergo a reversible transition when titrated over the pH range of 7.00-9.70. This transition produces a conversion from a pentacoordinate high-spin to a hexacoordinate low-spin heme active site and is clearly apparent in the heme optical absorption spectra. Here, we report the characterization of this transition and its effect upon the local heme environment using various optical spectroscopies. The formation of hexacoordinate low-spin heme is interpreted to involve the binding of His-52 at the distal site after the perturbation of the extensive H-bonded network within and around the heme pocket of CCP(II) at alkaline pH. Interestingly, CD investigations of CCP(II) in the far-UV and Soret regions indicate the dissappearance of a single high-spin species and the existence of at least two low-spin species of CCP(II) as the pH is raised above 7.90. Furthermore, transient resonance Raman experiments demonstrate that the hexacoordinate low-spin species can be photolyzed within 10-ns laser pulses, producing a species similar to the low-pH (high-spin) form of CCP(II) at alkaline pH. However, the extent of photolysis is quite pH dependent, with a maximum photodissociation yield at pH = 8.50.

show abstract

Section: T H I S C O N T E N T Isupporting

confidence: 88%

Section: T H I S C O N T E N T Isupporting

confidence: 88%

Formation and photolability of low-spin ferrous cytochrome c peroxidase at alkaline pH

Wang¹,

Boldt²,

Ondrias³

1992

Biochemistry

View full text Add to dashboard Cite

show abstract

“…All CD spectra in the backbone region (210-240 nm; UV/CD) were recorded in a 0.05-cm path-length cell (∼150 µL) using a scan speed of 100 nm/min with a response time of 0.25 s. The UV/CD spectra reported here are the average of five scans at 0.2nm resolution and a bandwidth of 1 nm. The observed ellipticity θ (millidegrees) was divided by (10 × C × l) to convert to molar ellipticity [θ], where C is the concentration (moles per liter) and l the path-length (centimeters) (Sievers, 1978). For the CD spectra in the aromatic and Soret regions (240-480 nm), a 0.5-cm path-length cell was used at a protein concentration of ∼10 µM.…”

Section: Denaturation Of Ccp(mi) and Hrp As Monitored Bymentioning

confidence: 99%

Conformational States in Denaturants of Cytochrome c and Horseradish Peroxidases Examined by Fluorescence and Circular Dichroism

1998

View full text Add to dashboard Cite

Steady-state fluorescence and circular dichroism (CD) were used to examine the unfolding in denaturants of recombinant cytochrome c peroxidase [CCP(MI)] and horseradish peroxidase (HRP) in their ferric forms. CCP(MI) unfolds in urea and in guanidine hydrochloride (GdHCl) at pH 7.0, while HRP loses its secondary structure only in the presence of GdHCl. CCP(MI) unfolds in urea by two distinct steps as monitored by fluorescence, but the loss of its secondary structure as monitored by UV/CD occurs in a single step between 3.4 and 5 M urea and 1.5 and 2.5 M GdHCl. The localized changes detected by fluorescence involve the CCP(MI) heme cavity since the Soret maximum red-shifts from 408 to 416 nm, and the heme CD changes examined in urea are biphasic. The polypeptide of HRP also loses secondary structure in a single step between 1.2 and 2.7 M GdHCl as monitored by UV/CD, and a fluorescence-monitored transition involving conformational change in the Trp117-containing loop occurs above 4 M GdHCl. Free energies of denaturation extrapolated to 0 M denaturant (delta Gd,aq) of approximately 6 and approximately 4 kcal/mol were calculated for CCP(MI) and HRP, respectively, from the UV/CD data. The refolding mechanisms of the two peroxidases differ since heme capture in CCP(MI) is synchronous with refolding while apoHRP captures heme after refolding. Thus, the denatured form of apoHRP does not recognize heme and has to correctly refold prior to heme capture. The half-life for unfolding of native HRP in 6 M GdHCl is slow (519 s) compared to that for CCP(MI) (14.3 s), indicating that HRP is kinetically much more stable than CCP(MI). Treatment with EDTA and DTT greatly destabilizes HRP, and unfolding in 4 M GdHCl occurs with t1/2 = 0.42 s.

show abstract

“…This is a result of the CN-ligation, and has also been observed in the CNadducts of Ps. aeruginosa (Ellfolk et al., 1984a), horseradish (Keilin and Hartree 1951) and yeast cytochrome c peroxidases (Sievers, 1978).…”

Section: Cntitration Of Cytochrome C Peroxidasementioning

confidence: 99%

Spectroscopic characterization of cytochrome c peroxidase from Paracoccus denitrificans

Gilmour

Goodhew

Pettigrew

et al. 1993

Biochemical Journal

View full text Add to dashboard Cite

The cytochrome c peroxidase of Paracoccus denitrificans is similar to the well-studied enzyme from Pseudomonas aeruginosa. Like the Pseudomonas enzyme, the Paracoccus peroxidase contains two haem c groups, one high potential and one low potential. The high-potential haem acts as a source of the second electron for H2O2 reduction, and the low-potential haem acts as a peroxidatic centre. Reduction with ascorbate of the high-potential haem of the Paracoccus enzyme results in a switch of the low-potential haem to a high-spin state, as shown by visible and n.m.r. spectroscopy. This high-spin haem of the mixed-valence enzyme is accessible to ligands and binds CN- with a KD of 5 microM. The Paracoccus enzyme is significantly different from that from Pseudomonas in the time course of high-spin formation after reduction of the high-potential haem, and in the requirement for bivalent cations. Reduction with 1 mM ascorbate at pH 6 is complete within 2 min, and this is followed by a slow appearance of the high-spin state with a half-time of 10 min. Thus the process of reduction and spin state change can be easily separated in time and the intermediate form obtained. This separation is also evident in e.p.r. spectra, although the slow change involves an alteration in the low-spin ligation at this temperature rather than a change in spin state. The separation is even more striking at pH 7.5, where no high-spin form is obtained until 1 mM Ca2+ is added to the mixed-valence enzyme. The spin-state switch of the low-potential haem shifts the midpoint redox potential of the high-potential haem by 50 mV, a further indication of haem-haem interaction.

show abstract

Circular dichroism studies on cytochrome peroxidase from baker's yeast (Saccharomyces cerevisiae)

Cited by 23 publications

References 50 publications

Formation and photolability of low-spin ferrous cytochrome c peroxidase at alkaline pH

Formation and photolability of low-spin ferrous cytochrome c peroxidase at alkaline pH

Conformational States in Denaturants of Cytochrome c and Horseradish Peroxidases Examined by Fluorescence and Circular Dichroism

Spectroscopic characterization of cytochrome c peroxidase from Paracoccus denitrificans

Contact Info

Product

Resources

About