scholarly journals Redox-linked spin-state changes in the di-haem cytochrome c-551 peroxidase from Pseudomonas aeruginosa

1985 ◽  
Vol 230 (1) ◽  
pp. 227-237 ◽  
Author(s):  
N Foote ◽  
J Peterson ◽  
P M Gadsby ◽  
C Greenwood ◽  
A J Thomson
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cytochrome C ◽  
High Spin ◽  
Spin State ◽  
High Potential ◽  
Reduced Form ◽  
Inactive Form ◽  
Temperature Spectra ◽  
State Changes ◽  
Haem Protein

Magnetic-c.d., e.p.r. and optical-absorption spectra are reported for the half-reduced form of Pseudomonas aeruginosa cytochrome c-551 peroxidase, a di-haem protein, and its fluoride derivative. Comparison of this enzyme species with oxidized peroxidase shows the occurrence of spin-state changes at both haem sites. The high-potential haem changes its state from partially high-spin to low-spin upon reduction. This is linked to a structural alteration at the ferric low-potential haem group, causing it to change from low-spin to high-spin. Low-temperature spectra demonstrate photolysis of an endogenous ligand of the high-potential haem. In addition, an inactive form of enzyme is examined in which the structural change at the ferric low-potential haem does not occur on reduction of the high-potential haem.

scholarly journals Spectroscopic characterization of cytochrome c peroxidase from Paracoccus denitrificans

1993 ◽  
Vol 294 (3) ◽  
pp. 745-752 ◽  
Author(s):  
R Gilmour ◽  
C F Goodhew ◽  
G W Pettigrew ◽  
S Prazeres ◽  
I Moura ◽  
...  
Keyword(s):  
Cytochrome C ◽  
High Spin ◽  
Spin State ◽  
Time Course ◽  
Paracoccus Denitrificans ◽  
Mixed Valence ◽  
Cytochrome C Peroxidase ◽  
High Spin State ◽  
High Potential ◽  
Intermediate Form

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.

scholarly journals Cytochrome c″ isolated from Methylophilus methylotrophus. An example of bis-histidine-co-ordinated Fe3+ haem, with near-perpendicular orientation of the ligands

1990 ◽  
Vol 270 (2) ◽  
pp. 413-417 ◽  
Author(s):  
M J Berry ◽  
S J George ◽  
A J Thomson ◽  
H Santos ◽  
D L Turner
Keyword(s):  
Low Temperature ◽  
Cytochrome C ◽  
High Spin ◽  
Magnetic Circular Dichroism ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Water Soluble ◽  
Reduced Form ◽  
Resonance Spectroscopy ◽  
Methylophilus Methylotrophus ◽  
Perpendicular Orientation

Cytochrome c″ (Methylophilus methylotrophus) is a soluble protein, Mr 15,000, possessing one haem which is high-spin in the reduced state but switches to a low-spin form on oxidation. Low-temperature electron-paramagnetic-resonance spectroscopy of the oxidized state shows a low-spin signal at gz = 3.65 with a folded line-shape typical of a haem of low rhombicity, and the near-infrared magnetic-circular-dichroism (m.c.d.) spectra reveal an unusually intense (delta epsilon = 400 M-1.cm-1 at 5 T, 4.2 K) charge-transfer band at 1560 nm, establishing that the oxidized haem is co-ordinated by two His residues in a near-perpendicular orientation. This conformation is well established for transmembrane b cytochromes, but this appears to be the first example in a water-soluble cytochrome. The low-temperature m.c.d. spectra of the reduced form of the protein confirms that the haem contains a high-spin Fe2+ ligated by one His residue. The redox-linked spin-state change releases a His group. Since this residue is likely to bind a proton at pH values less than 6.5, this cytochrome may provide a useful model of a molecular mechanism of a redox-linked proton uptake and release process.

scholarly journals The nature of species prepared by photolysis of half-reduced, fully reduced and fully reduced carbonmonoxy-cytochrome c-551 peroxidase from Pseudomonas aeruginosa

1984 ◽  
Vol 223 (2) ◽  
pp. 379-391 ◽  
Author(s):  
C Greenwood ◽  
N Foote ◽  
J Peterson ◽  
A J Thomson
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Fine Structure ◽  
Low Temperature ◽  
Cytochrome C ◽  
Activation Barrier ◽  
Magnetic Circular Dichroism ◽  
High Potential ◽  
Quantum Mechanical ◽  
Methionine Residue ◽  
Magnetic Circular Dichroism Spectroscopy

The half-reduced, fully reduced and fully reduced CO-bound forms of the enzyme cytochrome c-551 peroxidase isolated from Pseudomonas aeruginosa were examined by a combination of low-temperature absorption and magnetic-circular-dichroism spectroscopy. Deliberate low-temperature (4.2K) photolysis of these forms of the enzyme, in all of which the high-potential haem is in the ferrous state, revealed that this haem group, assigned to have a histidine-methionine ligand set, is photosensitive. The photolabile ligand is most likely to be the methionine residue, and the product of photolysis, namely the high-spin (S = 2) ferrous form, is stable at low temperature (4.2K). Warming to approx. 20K allows thermal recombination to occur, restoring the low-spin (S = 0) state. The low-potential haem (bis-histidine ligation) is photoinert in both ferric and ferrous states; however, the photosensitive CO adduct of this centre cannot be maintained as the photolysed (S = 2) product at 4.2K. This surprising observation may be due to quantum-mechanical tunnelling of the CO through the activation barrier even at 4.2K, implying that the activation barrier to thermal recombination is both narrow and low. Low-temperature absorption spectroscopy reveals that the high-potential haem has a very characteristic low-spin ferrous spectrum with intense highly structured beta- and split alpha-bands, whereas the spectrum of the low-potential ferrous haem contains alpha- and beta-bands devoid of fine structure.

scholarly journals Cytochrome c-551 and azurin oxidation catalysed by Pseudomonas aeruginosa cytochrome oxidase. A steady-state kinetic study

1985 ◽  
Vol 230 (3) ◽  
pp. 797-805 ◽  
Author(s):  
M G Tordi ◽  
M C Silvestrini ◽  
A Colosimo ◽  
L Tuttobello ◽  
M Brunori
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Steady State ◽  
Cytochrome C ◽  
Cytochrome Oxidase ◽  
Binding Sites ◽  
Time Course ◽  
Product Inhibition ◽  
Inactive Form ◽  
Catalytically Active ◽  
The Stability

The kinetics of oxidation of azurin and cytochrome c-551 catalysed by Pseudomonas aeruginosa cytochrome oxidase were re-investigated, and the steady-state parameters were evaluated by parametric and non-parametric methods. At low concentrations of substrates (e.g. less than or equal to 50 microM) the values obtained for Km and catalytic-centre activity are respectively 15 +/- 3 microM and 77 +/- 6 min-1 for azurin and 2.15 +/- 0.23 microM and 66 +/- 2 min-1 for cytochrome c-551, in general accord with previous reports assigning to cytochrome c-551 the higher affinity for the enzyme and to azurin a slightly higher catalytic rate. However, when the cytochrome c-551 concentration was extended well beyond the value of Km, the initial velocity increased, and eventually almost doubled at a substrate concentration greater than or equal to 100 microM. This result suggests a ‘half-hearted’ behaviour, since at relatively low cytochrome c-551 concentrations only one of the two identical binding sites of the dimeric enzyme seems to be catalytically active, possibly because of unfavourable interactions influencing the stability of the Michaelis-Menten complex at the second site. When reduced azurin and cytochrome c-551 are simultaneously exposed to Ps. aeruginosa cytochrome oxidase, the observed steady-state oxidation kinetics are complex, as expected in view of the rapid electron transfer between cytochrome c-551 and azurin in the free state. In spite of this complexity, it seems likely that a mechanism involving a simple competition between the two substrates for the same active site on the enzyme is operative. Addition of a chemically modified and redox inactive form of azurin (Hg-azurin) had no effect on the initial rate of oxidation of either azurin and cytochrome c-551, but clearly altered the time course of the overall process by removing, at least partially, the product inhibition. The results lead to the following conclusions: (i) reduced azurin and cytochrome c-551 bind at the same site on the enzyme, and thus compete; (ii) Hg-azurin binds at a regulatory site, competing with the product rather than the substrate; (iii) the two binding sites on the dimeric enzyme, though intrinsically equivalent, display unfavourable interactions. Since water is the product of the reduction of oxygen, point (iii) has important implications for the reaction mechanism.

Structure of cytochrome c′: a dimeric, high-spin haem protein

Nature ◽  
1980 ◽  
Vol 286 (5770) ◽  
pp. 302-304 ◽  
Author(s):  
Patricia C. Weber ◽  
R. G. Bartsch ◽  
M. A. Cusanovich ◽  
R. C. Hamlin ◽  
A. Howard ◽  
...  
Keyword(s):  
Cytochrome C ◽  
High Spin ◽  
Haem Protein

Cytochromes of the Purple Sulfur Bacterium Ectothiorhodospira shaposhnikovii

1984 ◽  
Vol 39 (9-10) ◽  
pp. 894-901 ◽  
Author(s):  
Werner H. Kusche ◽  
Hans G. Trüper
Keyword(s):  
Molecular Weight ◽  
Cytochrome C ◽  
Redox Potential ◽  
Cytochrome B ◽  
High Spin ◽  
Isoelectric Point ◽  
Sodium Dodecylsulfate ◽  
Reduced Form ◽  
Reduced Sulfur Compounds ◽  
Purple Sulfur Bacterium

Abstract Two c-type cytochromes (a high spin cytochrome c′ and a low spin cytochrome c-553(549) with asymmetrical α-band) and a low spin cytochrome b-558 from the purple sulfur bacterium Ectothiorhodospira shaposhnikovii were purified by ion exchange chromatography and gel filtration and characterized. Cytochrome c′ has a molecular weight of 33000 (determined by sodium dodecylsulfate electrophoresis), an isoelectric point at pH 4.5 and a redox potential of +37 mV. Absorption spectra show in the oxidized state maxima at 404 nm and in the range of 635 nm, in the reduced form maxima at 426.5 nm, 549 nm and a shoulder at 435 nm. The best purity index obtained was 0.48 (A280/A426.5). Reduced cytochrome c′ reacts with carbon monoxide. Cytochrome c-553(549) has a molecular weight of 10400, an isoelectric point at pH 5.1 and a redox potential of +248 mV. The oxidized form shows the Soret-band at 410 nm. The reduced protein reveals an asymmetrical a-band at 553 nm with a shoulder at 549 nm, the a-band at 522 nm with a shoulder at 528 nm and the γ-band at 416 nm. The best purity index obtained was 0.18 (A280/A416). Roth cytochromes could be isolated from the soluble fraction as well as from Triton X-100 treated membranes. Furthermore very low amounts of cytochromes c-553 and c-552.5 could be detected in detergent treated chromatophores. Cytochrome 6-558 - obtained from cells grown in the presence of reduced sulfur compounds in the medium - seems to be soluble or only weakly bound to the membrane. It has a molecular weight of 15800. an isoelectric point at pH 4.1 and a redox potential of -210 mV. The hemoprotein shows absorption maxima at 424.5 nm. 526.5 nm and 556.5 nm in the reduced form and at 416 nm in the oxidized state. The best purity index obtained was 0.26 (A280/A42,4.5). In addition, there were hints for the occurrence of a high spin cytochrome b′. The cytochrome pattern as well as the amount of cytochromes were dependent on growth conditions.

scholarly journals The kinetics of the oxidation of cytochrome c by Paracoccus cytochrome c peroxidase

1994 ◽  
Vol 300 (3) ◽  
pp. 907-914 ◽  
Author(s):  
R Gilmour ◽  
C F Goodhew ◽  
G W Pettigrew ◽  
S Prazeres ◽  
J J Moura ◽  
...  
Keyword(s):  
Cytochrome C ◽  
High Spin ◽  
Spin State ◽  
Paracoccus Denitrificans ◽  
Cytochrome C Peroxidase ◽  
High Spin State ◽  
Enzyme Activation ◽  
First Order ◽  
Ferrocytochrome C ◽  
Kinetics Of

In work that is complementary to our investigation of the spectroscopic features of the cytochrome c peroxidase from Paracoccus denitrificans [Gilmour, Goodhew, Pettigrew, Prazeres, Moura and Moura (1993) Biochem. J. 294, 745-752], we have studied the kinetics of oxidation of cytochrome c by this enzyme. The enzyme, as isolated, is in the fully oxidized form and is relatively inactive. Reduction of the high-potential haem at pH 6 with ascorbate results in partial activation of the enzyme. Full activation is achieved by addition of 1 mM CaCl2. Enzyme activation is associated with formation of a high-spin state at the oxidized low-potential haem. EGTA treatment of the oxidized enzyme prevents activation after reduction with ascorbate, while treatment with EGTA of the reduced, partially activated, form abolishes the activity. We conclude that the active enzyme is a mixed-valence form with the low-potential haem in a high-spin state that is stabilized by Ca2+. Dilution of the enzyme results in a progressive loss of activity, the extent of which depends on the degree of dilution. Most of the activity lost upon dilution can be recovered after reconcentration. The M(r) of the enzyme on molecular-exclusion chromatography is concentration-dependent, with a shift to lower values at lower concentrations. Values of M(r) obtained are intermediate between those of a monomer (39,565) and a dimer. We propose that the active form of the enzyme is a dimer which dissociates at high dilution to give inactive monomers. From the activity of the enzyme at different dilutions, a KD of 0.8 microM can be calculated for the monomerdimer equilibrium. The cytochrome c peroxidase oxidizes horse ferrocytochrome c with first-order kinetics, even at high ferrocytochrome c concentrations. The maximal catalytic-centre activity (‘turnover number’) under the assay conditions used is 62,000 min-1, with a half-saturating ferrocytochrome c concentration of 3.3 microM. The corresponding values for the Paracoccus cytochrome c-550 (presumed to be the physiological substrate) are 85,000 min-1 and 13 microM. However, in this case, the kinetics deviate from first-order progress curves at all ferrocytochrome c concentrations. Consideration of the periplasmic environment in Paracoccus denitrificans leads us to propose that the enzyme will be present as the fully active dimer supplied with saturating ferrocytochrome c-550.

scholarly journals Two distinct binding sites for high potential iron-sulfur protein and cytochrome c on the reaction center-bound cytochrome of Rubrivivax gelatinosus.

2004 ◽  
Vol 279 (44) ◽  
pp. 46350
Author(s):  
Jean Alric ◽  
Makoto Yoshida ◽  
Kenji V.P. Nagashima ◽  
Rainer Hienerwadel ◽  
Pierre Parot ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Reaction Center ◽  
Binding Sites ◽  
High Potential ◽  
Iron Sulfur ◽  
Sulfur Protein ◽  
Iron Sulfur Protein ◽  
Rubrivivax Gelatinosus
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