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W J Ingledew

Publications and source records attributed to W J Ingledew.

At least 19 recordsLinked to original sources
PubMed2005

A study of the horseradish peroxidase catalytic site by FTIR spectroscopy.

Vibrational changes in the catalytic site of horseradish peroxidase were investigated by FTIR (Fourier-transform infrared) spectroscopy in the 1000-2500 cm(-1) range. Difference spectra were generated by photolysis of the haemII-CO compound at different pH/pD values. The spectra report on the fine structure around the catalytic site and show vibrational changes of protein backbone, amino acid residues and cofactors. Assignments of the FTIR vibrations can be made based upon known crystal structures, comparisons with absorption frequencies and extinction coefficients of model amino acids and cofactors, effects of H2O/2H2O exchange and changes of pH/pD. Concomitant with the photolysis of the CO ligand are changes due to haem and protein vibrations, predominant among which are arginine and histidine residue vibrations.

Armoracia↗
PubMed2000

Angular dependences of perpendicular and parallel mode electron paramagnetic resonance of oxidized beef heart cytochrome c oxidase.

Cytochrome c oxidase catalyzes the reduction of oxygen to water with a concomitant conservation of energy in the form of a transmembrane proton gradient. The enzyme has a catalytic site consisting of a binuclear center of a copper ion and a heme group. The spectroscopic parameters of this center are unusual. The origin of broad electron paramagnetic resonance (EPR) signals in the oxidized state at rather low resonant field, the so-called g' = 12 signal, has been a matter of debate for over 30 years. We have studied the angular dependence of this resonance in both parallel and perpendicular mode X-band EPR in oriented multilayers containing cytochrome c oxidase to resolve the assignment. The "slow" form and compounds formed by the addition of formate and fluoride to the oxidized enzyme display these resonances, which result from transitions between states of an integer-spin multiplet arising from magnetic exchange coupling between the five unpaired electrons of high spin Fe(III) heme a(3) and the single unpaired electron of Cu(B). The first successful simulation of similar signals observed in both perpendicular and parallel mode X-band EPR spectra in frozen aqueous solution of the fluoride compound of the closely related enzyme, quinol oxidase or cytochrome bo(3), has been reported recently (Oganesyan et al., 1998, J. Am. Chem. Soc. 120:4232-4233). This suggested that the exchange interaction between the two metal ions of the binuclear center is very weak (|J| approximately 1 cm(-1)), with the axial zero-field splitting (D approximately 5 cm(-1)) of the high-spin heme dominating the form of the ground state. We show that this model accounts well for the angular dependences of the X-band EPR spectra in both perpendicular and parallel modes of oriented multilayers of cytochrome c oxidase derivatives and that the experimental results are inconsistent with earlier schemes that use exchange coupling parameters of several hundred wavenumbers.

Animals↗
PubMed2000

ENDOR spectroscopic studies of stable semiquinone radicals bound to the Escherichia coli cytochrome bo3 quinol oxidase.

The putative oxidation of ubiquinol by the cytochrome bo3 terminal oxidase of Escherichia coli in sequential one-electron steps requires stabilization of the semiquinone. ENDOR spectroscopy has recently been used to study the native ubisemiquinone radical formed in the cytochrome bo3 quinone-binding site [Veselov, A.V., Osborne, J.P., Gennis, R.B. & Scholes, C.P. (2000) Biochemistry 39, 3169-3175]. Comparison of these spectra with those from the decyl-ubisemiquinone radical in vitro indicated that the protein induced large changes in the electronic structure of the ubisemiquinone radical. We have used quinone-substitution experiments to obtain ENDOR spectra of ubisemiquinone, phyllosemiquinone and plastosemiquinone anion radicals bound at the cytochrome bo3 quinone-binding site. Large changes in the electronic structures of these semiquinone anion radicals are induced on binding to the cytochrome bo3 oxidase. The changes in electronic structure are, however, independent of the electronic structures of these semiquinones in vitro. Thus it is shown to be the structure of this binding site in the protein, not the covalent structure of the bound quinone, that determines the electronic structure of the protein-bound semiquinone.

Cytochrome b Group↗
PubMed1998

Identification of a stable semiquinone intermediate in the purified and membrane bound ubiquinol oxidase-cytochrome bd from Escherichia coli.

The quinol oxidase, cytochrome bd, functions as a terminal oxidase in the Escherichia coli respiratory chain, reducing O2 to water and using ubiquinol-8 or menaquinol-8 as its immediate reductant. The oxidation of quinol is by the low-spin ferri-haem, cytochrome b558. This occurs at a quinol-binding site by sequential one electron steps, requiring the stabilisation of the semiquinone intermediate. We have observed, by EPR spectroscopy, the properties of this semiquinone radical in appropriately poised samples of purified enzyme reconstituted with excess of ubiquinone-8 and menaquinone-8 analogues. The line width of the EPR spectrum is approximately 0.9 mT, which is consistent with a semiquinone anion of this type. The line shape is Gaussian. The semiquinone is highly stabilised with respect to free semiquinone; significant free radical can be observed at pH 7.0 and above. The pH dependence of the redox reactions indicate that the anionic form of the semiquinone and the neutral form of the quinol predominate in the pH range studied. The pH dependence of the mid-point potentials of the one electron reactions from pH 7.0-9.0 is 120 mV/pH change for the semiquinone anion to quinol (E2) and none for the quinone to semiquinone (E1). The semiquinone radical is attenuated on titration with putative inhibitors of this quinone-binding site. We have similarly studied the semiquinone in membrane preparations from a strain with overexpression of cytochrome bd oxidase. The data can be fitted with the assumption of a single quinone-binding site.

Bacterial Proteins↗
PubMed1998

Structure determination of a 16.8 kDa copper protein at 2.1 A resolution using anomalous scattering data with direct methods.

The structure of rusticyanin, an acid-stable copper protein, has been determined at 2.1 A resolution by direct methods combined with the single-wavelength anomalous scattering (SAS) of copper (f" = 3.9 e-) and then conventionally refined (Rcryst = 18.7%, Rfree = 21.9%). This is the largest unknown protein structure (Mr approximately /= 16.8 kDa) to be determined using the SAS and direct-methods approach and demonstrates that by exploiting the anomalous signal at a single wavelength, direct methods can be used to determine phases at typical (approximately 2 A) macromolecular crystallographic resolutions. Extrapolating from the size of the anomalous signal for copper (f" approximately 4 e-), this result suggests that the approach could be used for proteins with molecular weights of up to 33 kDa per Se (f"max++ = 8 e- at the 'white line') and 80 kDa for a Pt derivative (f"max = 19 e- at the 'white line', L3 edge). The method provides a powerful alternative in solving a de novo protein structure without either preparing multiple crystals (i.e. isomorphous heavy-atom derivative plus native crystals) or collecting multi-wavelength anomalous diffraction (MAD) data.

Azurin↗
PubMed1998

Angular dependence of electron paramagnetic resonances of an azide-NO complex of cytochrome c oxidase: orientation of the haem-copper axis in cytochrome aa3 from ox heart.

The orientation dependence of the EPR signals arising from the azide-nitric oxide complex of cytochrome oxidase was investigated using oriented multilayers of mitochondrial membranes from ox heart. Variations in line shape of the DeltaMS=1 signal of the triplet state were apparent, whilst the DeltaMS=2 transitions between g=4.7 and 3.9 varied in intensity as the angle of the applied magnetic field was varied. These half-field signals were maximal with the field parallel to the membrane plane. A model of the bi-liganded azide-nitric oxide complex has been constructed, in which the nitric oxide is bound to the high-spin haem in a bent configuration, with the Fe-N=O plane at 60-90 degrees to the membrane plane and the azide bound to the copper, distal from the haem. In addition, angular variations of the signals at g'=11 and g' around 3.5, derived from an integer-spin complex, were also observed.

Animals↗
PubMed1997

EPR spectroscopy of Escherichia coli cytochrome bo which lacks CuB.

The spectroscopic and ligand-binding properties of a copper-deficient cytochrome bo3, a member of the haem-copper superfamily of terminal oxidases, are reported and contrasted with those of the native enzyme. The enzyme lacks the copper atom (CuB) which is normally an integral part of the catalytic site. The consequences of loss of the CuB are the loss of antiferromagnetic coupling to the high-spin haem and an inability to form any of the integer-spin derivatives of the enzyme. Low-spin compounds of the normally high-spin haem are still formed with appropriate ligands, although these are modified.

Azides↗
PubMed1996

New insights into the catalytic cycle of flavocytochrome b2.

Flavocytochrome b2 from Saccharomyces cerevisiae couples L-lactate dehydrogenation to cytochrome c reduction in the mitochondrial intermembrane space. The catalytic cycle for this process can be described in terms of five consecutive electron-transfer events. L-Lactate dehydrogenation results in the two-electron reduction of FMN. The two electrons are individually passed to b2-heme (intramolecular electron transfer) and then onto cytochrome c (intermolecular electron transfer). At 25 degrees C, I 0.10, in the presence of saturating concentrations of ferricytochrome c and L-lactate, the catalytic cycle progresses with rate constant 104 (+/- 5) s-1 [per L-lactate oxidized; Miles, C. S., Rouviere-Fourmy, N., Lederer, F., Mathews, F. S., Reid, G. A., & Chapman, S. K. (1992) Biochem. J. 285, 187-192]. Stopped-flow spectrophotometry has been used to show that the major rate-limiting step in the catalytic cycle is electron transfer from flavin semiquinone to b2-heme. This conclusion is based on the observation that pre-steady-state flavin oxidation by ferricytochrome c takes place at 120 s-1. Although flavin oxidation involves several other electron transfer steps, these are considered too fast to contribute significantly to the rate constant. It was also shown that the reaction product, pyruvate, is able to inhibit pre-steady-state flavin oxidation (Ki = 40 +/- 17 mM) consistent with reports that it acts as a noncompetitive inhibitor in the steady state at high concentrations [Ki = 30 mM; Lederer, F. (1978) Eur. J. Biochem, 88, 425-431]. This novel way of measuring the electron transfer rate constant is directly applicable to the catalytic cycle and has enabled us to derive a self-consistent model for it, based also on data collected for enzyme reduction [Miles, C. S., Rouviere-Fourmy, N., Lederer, F., Mathews, F. S., Reid, G. A., & Chapman, S. K. (1992) Biochem. J. 285, 187-192] and its interaction with cytochrome c [Daff, S., Sharp, R. E., Short, D. M., Bell, C., White, P., Manson, F. D. C., Reid, G. A., & Chapman, S. K. (1996) Biochemistry 35, 6351-6357]. Rapid-freezing quenched-flow EPR has been used to confirm the model by demonstrating that during steady-state turnover of the enzyme approximately 75% of the flavin is in the semiquinone oxidation state.

Catalysis↗
PubMed1996

The structural gene for rusticyanin from Thiobacillus ferrooxidans: cloning and sequencing of the rusticyanin gene.

The rusticyanin gene from the acidophilic chemolithotroph Thiobacillus ferrooxidans has been cloned and sequenced. A central portion of the gene was identified by PCR reactions utilising primers optimised for codon bias followed by nested PCR with degenerate primers. The 5' and 3' ends of the rusticyanin gene were then cloned using degenerate primers to each end and anchor sequences to the known internal sequence. The entire gene was amplified using Tli DNA polymerase and specific primers to the 5' and 3' ends and the sequence confirmed after cloning into Bluescript and transformation of XL-1 Blue Escherichia coli.

Amino Acid Sequence↗
PubMed1996

ATP-dependent copper transporter, in the Golgi apparatus of rat hepatocytes, transports Cu(II) not Cu(I).

The Wilson disease adenosinetriphosphatase (ATPase; ATP7B) is believed to bind copper as Cu(I). We provide evidence to suggest that the ATPase actually transports Cu as Cu(II). When the copper is presented to rat liver microsomes as Cu(I), virtually all uptake is ATP independent. If the copper is presented as copper oxalate [Cu(II)], total uptake is reduced to approximately 10% of Cu(I) levels, but ATP-dependent uptake rises, both as a proportion of total uptake and in absolute terms. The reducing agent vitamin C and the Cu(I) chelator bathocuproine both override the effect of oxalate. The data indicate that there are two transporters in the microsomes, an ATP-independent Cu(I) transporter and an ATP-dependent Cu(II) pump. The activity of the Cu(I) transporter correlates most strongly with alkaline phosphatase, suggesting that it is derived from plasma membrane contamination. Cu(II) ATP-dependent transport correlates only with beta-1, 4-galactosyltransferase, which indicates that it is located in the Golgi apparatus.

Adenosine Triphosphatases↗
PubMed1995

X-ray absorption studies and homology modeling define the structural features that specify the nature of the copper site in rusticyanin.

Rusticyanin, a blue copper protein, possessing the highest redox potential among this class of proteins and a high stability at acidic pH reveals homology with the C-terminal end of the other single copper containing blue proteins and an interesting homology to parts of the blue copper domain of the multi-copper proteins such as the nitrite reductases. Extended X-ray absorption fine structure (EXAFS) data at pH 2.0 reveal that Cu is ligated to two His and a Cys in the inner coordination sphere, similar to other blue copper centers. Modeling studies suggest that His85 is the ligating histidine from the N-terminal end. Its neighboring residue is a serine rather than the asparagine found in all known blue Cu proteins. The high stability of the copper site may arise in part due to this substitution. The Cu binding site is surrounded by aromatic residues which may provide further protection for the metal in an acidic environment. In addition, the high number of solvent-exposed uncompensated lysine residues is likely to be of functional relevance under low pH conditions. EXAFS data show a very small change (relative to azurin) in the copper site upon reduction, consistent with a more constrained copper center in rusticyanin compared to azurin and a higher redox potential.

Absorptiometry, Photon↗
PubMed1995

Studies on a stabilisation of ubisemiquinone by Escherichia coli quinol oxidase, cytochrome bo.

The Escherichia coli quinol oxidase, cytochrome bo, is closely related to the cytochrome c oxidase, cytochrome aa3 in all aspects of its structure and function except for the replacement of the cytochrome-c-binding site and its attendant CuA prosthetic group with a quinone-binding site. The putative oxidation of quinol by ferrihaem (cytochrome b) at this site in sequential one-electron steps requires the stabilisation of semiquinone. We have observed, by electron paramagnetic resonance, the properties of a ubisemiquinone radical in appropriately poised samples of purified enzyme reconstituted with excess ubiquinone. The ubisemiquinone is highly stabilised with respect to free ubisemiquinone; significant free radical can be observed even at pH 7.0, while at pH 9.0 the stability constant is 5-10. The pH dependence of the stability constant indicates that the anionic form of the semiquinone predominates above pH 7.5. The two-electron couple has an Em7 of approximately 70 mV. Below pH 9, the pH dependence of the two-electron couple is -60mV/pH, indicative of a 2H+/2e- reaction. The line width of the EPR spectrum is approximately 0.9 mT, which is consistent with a ubisemiquinone anion. In comparison with other respiratory chain Q.- species that have been described, the relaxation rate in the presence of reduced haems appears comparable to magnetically isolated Q.- radicals. Partially resolved splittings of approximately 0.4 mT can be observed in the spectrum of Q.-bo (QH.bo).

Binding Sites↗
PubMed1994

Strong-field and integral spin-ligand complexes of the cytochrome bo quinol oxidase in Escherichia coli membrane preparations.

The cytochrome bo-type terminal oxidase of Escherichia coli is an analogue of mammalian aa3-type cytochrome c oxidase. The catalytic core of both enzymes is a binuclear site containing a penta-coordinate heme (heme o or a3) and copper (CuB). Herein we report on UV-visible and magnetic properties of ligand complexes of the binuclear site of cytochrome bo. Cyanide, sulfide, and azide react with the Fe(3+)-Cu+ center to give EPR-detectable low-spin complexes, analogous to those formed by cytochrome aa3. Analyses of the ligand fields of these complexes indicate that heme o has a single axial histidine ligand. Cyanide and azide react with the Fe(3+)-Cu2+ center to yield forms observable via UV-visible spectroscopy but not EPR. With formate and fluoride, cytochrome bo forms integral spin complexes similar to those of cytochrome aa3. These complexes have UV-visible characteristics of high-spin species, but EPR spectra show features which appear to correspond to transitions within an integral spin multiplet. Cytochrome bo forms another integral spin complex with azide and NO which is nearly identical to the azide-NO species in cytochrome aa3. This suggests that the binuclear centers of the two enzymes are quite similar.

Cell Membrane↗
PubMed1993

Site-directed mutants of the cytochrome bo ubiquinol oxidase of Escherichia coli: amino acid substitutions for two histidines that are putative CuB ligands.

The bo-type ubiquinol oxidase of Escherichia coli is a member of the superfamily of structurally related heme-copper respiratory oxidases. The members of this family, which also includes the aa3-type cytochrome c oxidases, contain at least two heme prosthetic groups, a six-coordinate low-spin heme, and a high-spin heme. The high-spin heme is magnetically coupled to a copper, CuB, forming a binuclear center which is the site of oxygen reduction to water. Vectorial proton translocation across the membrane bilayer appears to be another common feature of this superfamily of oxidases. It has been proposed previously that the two adjacent histidines in putative transmembrane helix VII (H333 and H334 in the E. coli sequence) of the largest subunit of the heme-copper oxidases are ligands to CuB. Previously reported mutagenesis studies of the E. coli bo-type oxidase and the aa3-type oxidase of Rhodobacter sphaeroides supported this model, as substitutions at these two positions produced nonfunctional enzymes but did not perturb the visible spectra of the two heme groups. In this work, six different amino acids, including potential copper-liganding residues, were substituted for H333 and H334 of the E. coli oxidase. All of the mutations resulted in inactive, but assembled, oxidase with both of the heme components present. However, cryogenic Fourier transform infrared (FTIR) spectroscopy of the CO adducts revealed that dramatic changes occur at the binuclear center as a result of each mutation and that CuB appears to be absent.(ABSTRACT TRUNCATED AT 250 WORDS)

Copper↗
PubMed1993

Ligand binding to the haem-copper binuclear catalytic site of cytochrome bo, a respiratory quinol oxidase from Escherichia coli.

The Escherichia coli quinol oxidase, cytochrome bo, is closely related to the cytochrome-c oxidase, cytochrome aa3 and reacts with ligands to the high-spin ferric haem or the high-spin ferric-cupric binuclear catalytic site in similar ways. Cyanide reacts with the isolated enzyme to give a low-spin complex, manifested by a red shift in the Soret band, the loss of an absorption band at 630 nm and the appearance of a low-spin ferric haem EPR resonance at g = 3.3. Sulphide also elicits a low-spin complex, whereas azide gives a mixture of low-spin and high-spin species. Formate and fluoride (and azide) give a blue shift in the Soret band and the development of a modified absorption band in the 600-650 nm range. These latter species are attributed to an integral spin compound involving the binuclear centre.

Anions↗