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 The optical properties of CuA in bovine cytochrome c oxidase determined by low-temperature magnetic-circular-dichroism spectroscopy

1983 ◽  
Vol 215 (2) ◽  
pp. 303-316 ◽  
Author(s):  
C Greenwood ◽  
B C Hill ◽  
D Barber ◽  
D G Eglinton ◽  
A J Thomson
Keyword(s):  
Circular Dichroism ◽  
Low Temperature ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Magnetic Circular Dichroism ◽  
Paramagnetic Species ◽  
Type I ◽  
Magnetization Curves ◽  
Magnetic Circular Dichroism Spectroscopy ◽  
Circular Dichroïsm

The visible-near-i.r.-region m.c.d. (magnetic-circular-dichroism) spectrum recorded at low temperature in the range 450-900 nm is reported for oxidized resting mammalian cytochrome c oxidase. M.c.d. magnetization curves determined at different wavelengths reveal the presence of two paramagnetic species. Curves at 576, 613 and 640 nm fit well to those expected for an x,y-polarized haem transition with g values of 3.03, 2.21 and 1.45, i.e. cytochrome a3+. The m.c.d. features at 515, 785 and 817 nm magnetize as a S = 1/2 paramagnet with average g values close to 2, and simulated m.c.d. magnetization curves obtained by using the observed g values of CuA2+, i.e. 2.18, 2.03 and 1.99, fit well to the experimental observations. The form of the m.c.d. magnetization curve at 466 nm is curious, but it can be explained if CuA2+ and cytochrome a3+ contribute with oppositely signed bands at this wavelength. By comparing the m.c.d. spectrum of the enzyme with that of extracted haem a-bisimidazole complex it has been possible to deconvolute the m.c.d. spectrum of CuA2+, which shows transitions throughout the spectral region from 450 to 950 nm. The m.c.d.-spectral properties of CuA2+ were compared with those of a well-defined type I blue copper centre in azurin isolated from Pseudomonas aeruginosa. The absolute intensities of the m.c.d. signals at equal fields and temperatures for CuA2+ are 10-20-fold greater than those for azurin. The optical spectrum of CuA2+ strongly suggests an assignment as a d9 ion rather than Cu(I) bound to a thiyl radical.

scholarly journals A study of the oxidized form of Pseudomonas aeruginosa cytochrome c-551 peroxidase with the use of magnetic circular dichroism

1984 ◽  
Vol 223 (2) ◽  
pp. 369-378 ◽  
Author(s):  
N Foote ◽  
J Peterson ◽  
P M A Gadsby ◽  
C Greenwood ◽  
A J Thomson
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Circular Dichroism ◽  
Cytochrome C ◽  
Room Temperature ◽  
Magnetic Circular Dichroism ◽  
Axial Ligand ◽  
Temperature Dependent ◽  
Magnetic Circular Dichroism Spectroscopy ◽  
Different Temperatures ◽  
Circular Dichroïsm

The magnetic properties at different temperatures of oxidized Pseudomonas aeruginosa cytochrome c-551 peroxidase were studied, with the use of the technique of magnetic-circular-dichroism spectroscopy. At 4.2K, both constituent haems were found to be low-spin, and the axial ligand pairs were identified as histidine-histidine and histidine-methionine. At room temperature high-spin signals were observed, amounting to less than 25% of the total haem present. These signals are concluded to arise mainly from a temperature-dependent spin-state equilibrium in the methionine-ligated haem.

scholarly journals The nature of haem a3 in the oxidized state of cytochrome c oxidase. Evidence from low-temperature magnetic-circular-dichroism spectroscopy in the near infrared region

1982 ◽  
Vol 207 (1) ◽  
pp. 167-170 ◽  
Author(s):  
A J Thomson ◽  
D G Englinton ◽  
B C Hill ◽  
C Greenwood
Keyword(s):  
Circular Dichroism ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Near Infrared ◽  
Magnetic Circular Dichroism ◽  
Infrared Region ◽  
Magnetic Circular Dichroism Spectroscopy ◽  
Near Infrared Region ◽  
Circular Dichroïsm ◽  
Inhibited Enzyme

The magnetic-circular-dichroism (m.c.d.) spectra of oxidized ‘resting’ bovine cytochrome c oxidase and the cyanide-inhibited form are reported at 5.15 T and at 4.2 K along with m.c.d. magnetization curves plotted at selected wavelengths. In both spectra there are features at 790nm and 1564nm due to Cua and haem a respectively, the e.p.r.-detectable components of the enzyme. There is a new peak at 1946nm only in the spectrum of the cyanide-inhibited enzyme. Arguments are advanced that assign this to low-spin ferric haem a3 bridged to Cua3, thereby forming a ferromagnetically coupled pair of metal ions.

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 Low-temperature magnetic-circular-dichroism spectroscopy of the iron-molybdenum cofactor and the complementary cofactor-less MoFe protein of Klebsiella pneumoniae nitrogenase

1984 ◽  
Vol 219 (2) ◽  
pp. 495-503 ◽  
Author(s):  
A E Robinson ◽  
A J M Richards ◽  
A J Thomson ◽  
T R Hawkes ◽  
B E Smith
Keyword(s):  
Low Temperature ◽  
Klebsiella Pneumoniae ◽  
Magnetic Circular Dichroism ◽  
Molybdenum Cofactor ◽  
Magnetization Curves ◽  
Mofe Protein ◽  
Paramagnetic Component ◽  
Magnetic Circular Dichroism Spectroscopy ◽  
Iron Molybdenum Cofactor ◽  
Circular Dichroïsm

The major metal clusters of the MoFe protein, Kpl, of Klebsiella pneumoniae nitrogenase were characterized separately by low-temperature magnetic-circular-dichroism spectroscopy. The spectra and magnetization curves of the extracted iron-molybdenum cofactor, FeMoco, and of ‘P’ clusters in NifB - Kpl, the inactive, FeMoco -less, MoFo protein from an nifB mutant, were measured and compared with those of the holoprotein. (When FeMoco and NifB - Kpl are combined, active Kpl is formed.) Reduced NifB - Kpl had a spectrum with a weak, paramagnetic, component superimposed on a diamagnetic background. The paramagnetic component was assigned to a contaminating, e.p.r.-active, species. Thionine-oxidized NifB - Kpl had a spectrum and magnetization properties very similar to those of thionine-oxidized Kpl, demonstrating that the ‘P’ clusters are not significantly affected by the absence of the FeMoco clusters. The spectra of reduced isolated FeMoco had similar magnetization curves but sharper features and higher intensities than those of this centre in dithionite-reduced Kpl . Furthermore, a shoulder near 580 nm in the Kpl spectrum was absent from that of FeMoco . This may be due to the loss of a ligand or to a change in symmetry of the FeMoco cluster on extraction.

scholarly journals Detection by low-temperature magnetic circular-dichroism spectroscopy of optical absorption bands due to molybdenum (V) in the form of xanthine oxidase giving the Desulpho Inhibited e.p.r. signal

1986 ◽  
Vol 233 (1) ◽  
pp. 107-110 ◽  
Author(s):  
J Peterson ◽  
C Godfrey ◽  
A J Thomson ◽  
G N George ◽  
R C Bray
Keyword(s):  
Circular Dichroism ◽  
Low Temperature ◽  
Xanthine Oxidase ◽  
Absorption Spectra ◽  
Magnetic Circular Dichroism ◽  
Electronic Absorption Spectra ◽  
Visible Region ◽  
Absorption Bands ◽  
Magnetic Circular Dichroism Spectroscopy ◽  
Circular Dichroïsm

The magnetic circular-dichroism (m.c.d.) spectra in the temperature range 1.5-100 K and the electronic absorption spectra at 4.2 and 295 K were measured for a number of desulpho xanthine oxidase derivatives. There were no significant differences between the absorption spectra that could be attributed to molybdenum. However, the visible-region m.c.d. spectrum of the ethanediol-treated metalloprotein (which gives rise to the Desulpho Inhibited e.p.r. signal) contained features assignable to Mo(V) absorption bands. This is the first report of the detection of optical bands of Mo(V) in an enzyme in the presence of other chromophoric centres.

scholarly journals Characterization of the partially reduced cyanide-inhibited derivative of cytochrome c oxidase by optical, electron-paramagnetic-resonance and magnetic-circular-dichroism spectroscopy

1981 ◽  
Vol 193 (3) ◽  
pp. 699-708 ◽  
Author(s):  
M K Johnson ◽  
D G Eglinton ◽  
P E Gooding ◽  
C Greenwood ◽  
A J Thomson
Keyword(s):  
Circular Dichroism ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Near Infrared ◽  
Magnetic Circular Dichroism ◽  
Circular Dichroism Spectroscopy ◽  
Paramagnetic Resonance ◽  
Magnetic Circular Dichroism Spectroscopy ◽  
Circular Dichroïsm ◽  
Reduced State

Optical. e.p.r. and near-infrared low-temperature m.c.d. (magnetic-circular-dichroism) spectroscopy were used to characterize the partially reduced cyanide-inhibited derivative of cytochrome c oxidase produced by anaerobic reductive titration with dithionite. The reductions of cytochrome a3+ and Cu2+a were followed by observation of the e.p.r. signals at g = 3.03, 2.21 and 1.5 and at g = 2.18, 2.03 and 1.99. As reduction proceeds new e.p.r. signals (g = 3.58 and 1.56) appear that quantify to give one haem per enzyme unit when a small excess of dithionite has been titrated in. The e.p.r. signal of the Cu2+a titrates in parallel with the disappearance of the band and 820nm in the optical absorption spectrum. The near-infrared m.c.d. spectrum shows the presence of the low-spin ferric haem, a3+, in the oxidized state of the enzyme, as a well-resolved positive peak at 1650nm. As reduction proceeds this band is replaced by one at 1550nm due to haem a3+(3)–CN in the partially reduced state. Hence as haem a3+(3)–CN becomes e.p.r.-detectable it also shows a near-infrared m.c.d. spectrum characteristic of a low-spin ferric haem. It is concluded that the partially reduced state of cyanide-inhibited cytochrome c oxidase contains a2+ . Cu+a . a3+(3)–CN . Cu+a3.

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