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R Aasa

Publications and source records attributed to R Aasa.

At least 37 records · Page 2Linked to original sources
PubMed1989

Analytical characterization of cytochrome oxidase preparations with regard to metal and phospholipid contents, peptide composition and catalytic activity.

A number of preparations of cytochrome oxidase have been analyzed for metals by energy dispersive X-ray fluorescence spectrometry. The EPR characteristics, the peptide compositions, the protein and phospholipid contents as well as the catalytic constants of the samples have also been determined. It is confirmed that the enzyme functional unit contains three copper atoms and one zinc atom in addition to two iron atoms. On the basis of the parameters determined for the different samples it is suggested that a high catalytic activity of a preparation can be correlated to a number of other analytical characteristics.

Copper↗
PubMed1987

Intermediates formed by the Co2+-activated ribulose-1,5-bisphosphate carboxylase/oxygenase from spinach studied by electron paramagnetic resonance spectroscopy.

Two enzyme-metal-bound intermediates formed by the Co2+-activated ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) have been studied by electron paramagnetic resonance (EPR) spectroscopy. Their rates of approach to a stationary state are different and their relative amounts at steady state are dependent on the concentration of ribulose 1,5-bisphosphate. It is therefore proposed that enzyme-metal-coordinated ribulose 1,5-bisphosphate and an enzyme-metal-coordinated enediolate anion of it, where bound ribulose 1,5-bisphosphate appears first, constitute the two EPR-detectable intermediates, respectively.

Cobalt↗
PubMed1987

EPR of azurins from Pseudomonas aeruginosa and Alcaligenes denitrificans demonstrates pH-dependence of the copper-site geometry in Pseudomonas aeruginosa protein.

The X- and Q-band EPR spectra of Pseudomonas aeruginosa (63Cu)azurin and Alcaligenes denitrificans azurin have been measured at pH = 5.2 and 9.2, in the presence and absence of 40% glycerol. The EPR spectra of both proteins could properly be simulated by taking into account a spread in the tetrahedral angle of the copper site. The change in the EPR spectrum of Pseudomonas aeruginosa (63Cu)azurin that is observed upon an increase of the pH from 5.2 to 9.2 is consistent with a small decrease of the average tetrahedral angle from 61 degrees to 60 degrees. This geometrical change is consistent with the interpretation of earlier NMR and EXAFS observations. No pH effect is observed for Alcaligenes denitrificans azurin, in agreement with predictions based on crystallographic evidence. Glycerol has only a marginal effect on the appearance of the EPR spectra, and does not alleviate the "g-strain."

Alcaligenes↗
PubMed1984

Cyanide inhibition of cytochrome c oxidase. A rapid-freeze e.p.r. investigation.

The inhibition of cytochrome c oxidase by cyanide, starting either with the resting or the pulsed enzyme, was studied by rapid-freeze quenching followed by quantitative e.p.r. It is found that a partial reduction of cytochrome oxidase by transfer of 2 electron equivalents from ferrocytochrome c to cytochrome a and CuA will induce a transition from a closed to an open enzyme conformation, rendering the cytochrome a3-CuB site accessible for cyanide binding, possibly as a bridging ligand. A heterogeneity in the enzyme is observed in that an e.p.r. signal from the cytochrome a3 3+-HCN complex is only found in 20% of the molecules, whereas the remaining cyanide-bound a3-CuB sites are e.p.r.-silent.

Binding Sites↗
PubMed1984

Anion binding to resting and half-reduced Pseudomonas cytochrome c peroxidase.

The anion-binding characteristics of resting and half-reduced Pseudomonas cytochrome c peroxidase (ferrocytochrome c-551: hydrogen peroxide oxidoreductase, EC 1.11.1.5) have been examined by EPR and optical spectroscopy with cyanide, azide and fluoride as ligands. The resting enzyme was found to be essentially inaccessible for ligation, which indicates that it has a closed conformation. In contrast, the half-reduced enzyme has a conformation in which the low-potential heme is easily accessible for ligands, a behavior parallel to that towards the substrate hydrogen peroxide (Rönnberg, M., Araiso, T., Ellfolk, N. and Dunford, H.B. (1981) Arch. Biochem. Biophys. 207, 197-204). Cyanide and azide caused distinct changes in the low-potential heme c moiety, and the gz values of the two low-spin derivatives were 3.14 and 3.22, respectively. Fluoride binds to the same heme, giving rise to a high-spin signal at g = 6. The dissociation constants of the anions differ widely from each other, the values for the cyanide, azide and fluoride being 23 microM, 2.5 mM and 0.13 M, respectively. In addition, a partial shift of the low-spin peak at g = 2.84 of the half-reduced species to 3.24 was observed even at low concentrations of fluoride.

Azides↗
PubMed1983

Properties and function of the two hemes in Pseudomonas cytochrome c peroxidase.

The oxidation-reduction potentials of the two c-type hemes of Pseudomonas aeruginosa cytochrome c peroxidase (ferrocytochrome c:hydrogen-peroxide oxidoreductase EC 1.11.1.5) have been determined and found to be widely different, about +320 and -330 mV, respectively. The EPR spectrum at temperatures below 77 K reveals only low-spin signals (gz 3.24 and 2.93), whereas optical spectra at room temperature indicate the presence of one high-spin and one low-spin heme in the enzyme. Optical absorption spectra of both resting and half-reduced enzyme at 77 K lack features of a high-spin compound. It is concluded that the heme ligand arrangement changes on cooling from 298 to 77 K with a concomitant change in the spin state. The active form of the peroxidase is the half-reduced enzyme, in which one heme is in the ferrous and the other in the ferric state (low-spin below 77 K with gz 2.84). Reaction of the half-reduced enzyme with hydrogen peroxide forms Compound I with the hemes predominantly in the ferric (gz 3.15) and the ferryl states. Compound I has a half-life of several seconds and is converted into Compound II apparently having a ferric-ferric structure, characterized by an EPR peak at g 3.6 with unusual temperature and relaxation behavior. Rapid-freeze experiments showed that Compound II is formed in a one-electron reduction of Compound I. The rates of formation of both compounds are consistent with the notion that they are involved in the catalytic cycle.

Cytochrome-c Peroxidase↗
PubMed1982

An investigation by e.p.r. and optical spectroscopy of cytochrome oxidase during turnover.

Cytochrome oxidase (EC 1.9.3.1; ferrocytochrome c:oxygen oxidoreductase) was studied during steady-state by optical and e.p.r. methods. Starting with either the 'resting' or the 'pulsed' enzyme, oxidase, cytochrome c, ascorbate and O2 were mixed and the reaction monitored optically. Tetramethylphenylenediamine was used as mediator to poise the steady-state to the desired reduction level. After mixing, the reaction was quenched by the used of rapid-freeze techniques. The e.p.r. spectra of samples captured at increasing tetramethylphenylenediamine concentrations (i.e. higher electron flux) show decreasing g = 2 (Cu A) and g = 3 (cytochrome a) signals. No Cu B or g = 6 signals (high-spin cytochrome a3) could be found during the reaction. Also, the signal with peaks at g = 1.69, 1.78 and 5 as well as the g = 12 signal was hardly detectable at higher turnover rates. The only new signal appearing during turnover is a radical signal, which is discussed in terms of a protein radical. Finally, a scheme is presented, proposing a catalytic cycle for cytochrome oxidase with respect to the O2 binding Cu B-cytochrome a3 unit.

Electron Spin Resonance Spectroscopy↗
PubMed1982

The structure of the paramagnetic oxygen intermediate in the cytochrome c oxidase reaction.

A paramagnetic intermediate with an unusual e.p.r. spectrum is formed when fully reduced cytochrome c oxidase is allowed to react with dioxygen at 173 K. The effect on the e.p.r. spectrum of using dioxygen enriched in 17O was investigated. These experiments show that an oxygen atom derived from dioxygen is bound to Cu2+ in the intermediate. The e.p.r. parameters can be explained in terms of a weak antiferromagnetic interaction (J approximately equal to 10 cm-1) between Cu2+B and cytochrome a3 in the low-spin ferryl ion state. It is suggested that an OH- ion bound to Cu2+B is hydrogen bonded to the oxygen atom of the ferryl ion in cytochrome a3.

Animals↗
PubMed1981

Electron paramagnetic resonance studies of Pseudomonas cytochrome c peroxidase.

The EPR spectrum at 15 K of Pseudomonas cytochrome c peroxidase, which contains two hemes per molecule, is in the totally ferric form characteristic of low-spin heme giving two sets of g-values with gz 3.26 and 2.94. These values indicate an imidazole-nitrogen : heme-iron : methionine-sulfur and an imidazole-nitrogen : heme-iron : imidazole-nitrogen hemochrome structure, respectively. The spectrum is essentially identical at pH 6.0 and 4.6 and shows only a very small amount of high-spin heme iron (g 5--6) also at 77 K. Interaction between the two hemes is shown to exist by experiments in which one heme is reduced. This induces a change of the EPR signal of the other (to gz 2.83, gy 2.35 and gx 1.54), indicative of the removal of a histidine proton from that heme, which is axially coordinated to two histidine residues. If hydrogen peroxide is added to the partially reduced protein, its EPR signal is replaced by still other signals (gz 3.5 and 3.15). Only a very small free radical peak could be observed consistent with earlier mechanistic proposals. Contrary to the EPR spectra recorded at low temperature, the optical absorption spectra of both totally oxidized and partially reduced enzyme reveal the presence of high-spin heme at room temperature. It seems that a transition of one of the heme c moieties from an essentially high-spin to a low-spin form takes place on cooling the enzyme from 298 to 15 K.

Azurin↗
PubMed1981

EPR spectroscopy of soybean lipoxygenase-1. Description and quantification of the high-spin fe(III) signals.

The interaction of soybean lipoxygenase-1 with 13-Ls-hydroperoxy-9-cis,11-trans-octadecadienoic acid, the product of the enzymic dioxygenation of linoleic acid, results in the formation of either a yellow or a purple coloured enzyme form depending on the amount of product used. The composition of the high-spin Fe(III) signals in the EPR spectra of both enzyme forms has been studied and the amount of EPR-visible iron determined by integration and simulation. Sets of g values of the species building up the high-spin Fe(III) signal around g 6 are derived from both third-order perturbation calculation and exact numerical diagonalization of the spin Hamiltonian describing the system. The results of these calculations are generally applicable to systems having S = 5/2. The iron in the native, colourless enzyme is almost EPR-nondetectable. The yellow form of the enzyme shows a complex EPR signal around g 6 which consists of contributions of at least three species with different ligand symmetry. The signal corresponds to approx. 75% of the total iron content. The g 6 signal of the purple Fe(III) enzyme corresponds roughly to the same amount of iron but the ratio between the different species is not the same as in the yellow enzyme. This enzyme form also shows an additional g 4.3 signal with a large amplitude but a relatively low integrated intensity (approx. 10% of the total iron content). The results are consistent with the suggested mechanism of the catalytic function of iron in lipoxygenase which was based on qualitative EPR results (De Groot, J.J.M.C., Veldink, G.A., Vliegenthart, J.F.G., Boldingh, J., Wever, R. and van Gelder, B.F. (1975) Biochim. Biophys. Acta 377, 71--79).

Electron Spin Resonance Spectroscopy↗
PubMed1980

EPR spectroscopy of soybean lipoxygenase-1. Determination of the zero-field splitting constants of high-spin Fe(III) signals from temperature and microwave frequency dependence.

The zero-field splitting constants (D) of the different components building up the high-spin Fe(III) EPR spectrum of lipoxygenase from soybeans were determined by two methods: (1) temperature dependence studies using the low-spin Fe(III) signal of cytochrome c at g 3 for accurate measuring of the temperature in the sample; (2) by establishing g-shift upon increasing the microwave frequency. The ranges of D for the axial and rhombic species contributing to the complex signal at g 6 are found to be 1.5-3.0 K and 1.8-4.4 K, respectively. The occurrence of such large ranges is attributed to variations in amount and number of species in the different samples. The combination of the applied methods offers a more generally applicable approach to the determination of zero-field splitting constants.

Cytochrome c Group↗
PubMed1980

A new copper(II) electron paramagnetic resonance signal in two laccases and in cytochrome c oxidase.

A new EPR signal from Cu2+ has been discovered in reductive experiments with type 2 copper-depleted laccase from Polyporus versicolor. A novel EPR signal has also been found in native laccase from Rhus vernicifera on oxidation of the reduced protein with H2O2. In reoxidation experiments with cytochrome c oxidase from beef heart, a new Cu2+ signal has been observed. With Rhus laccase, the new signal is shown to originate from one of the copper ions that are nondetectable in the resting enzyme, and evidence is presented for the signals in Polyporus laccase and cytochrome c oxidase also stemming from the metal pairs that are antiferromagnetically coupled in the oxidized enzymes. The new signals show strong rhombic character, and the EPR parameters place them in a category different from the signals of type 1 as well as of type 2 Cu2+ ions.

Basidiomycota↗
PubMed1980

Characterization of the low-temperature intermediates of the reaction of fully reduced soluble cytochrome oxidase with oxygen by electron-paramagnetic-resonance and optical spectroscopy.

The reaction of fully reduced soluble bovine heart cytochrome oxidase with O2 at 173K was investigated by low-temperature optical and e.p.r. spectroscopy, and the kinetics of the reaction were analysed by non-linear optimization techniques. The only e.p.r. signals seen during the course of the reaction are those attributable to low-spin cytochrome a3+ and CuA2+. Quantitative analysis of e.p.r. signals shows that, at the end point of the reaction at 173K, nearly 100% of CuA is in the cupric state but only about 40% of cytochrome a is in the ferric low-spin state. The optical spectra recorded at this stage of the reaction show incomplete oxidation of haem and the absence of a 655 nm absorption band. The only reaction scheme that accounts for both the e.p.r. and optical data is a four-intermediate mechanism involving a branching pathway. The reaction is initiated when fully reduced cytochrome oxidase reacts with O2 to form intermediate I. This is then converted into either intermediate IIA or intermediate IIB. Of these, intermediate IIB is a stable end product at 173 K, but intermediate IIA is converted into intermediate III, which is the stable state at 173 K in this branch of the mechanism. The kinetic analysis of the e.p.r. data allows the unambiguous assignments of the valence states of cytochrome a and CuA in the intermediates. Intermediate I contains cytochrome a2+ and CuA+, intermediate IIA contains low-spin cytochroma a3+ and CuA+, intermediate IIB contains cytochrome a2+ and CuA2+, and intermediate III contains low-spin cytochrome a3+ and CuA2+. The electronic state of the O2-binding CuBa3 couple during the reoxidation of cytochrome oxidase is discussed in terms of an integrated structure containing CuB, cytochrome a3 and O2.

Chemical Phenomena↗
PubMed1980

Characterization of the intermediates in the reaction of mixed-valence state soluble cytochrome oxidase with oxygen at low temperatures by optical and electron-paramagnetic-resonance spectroscopy.

The reaction of soluble mixed-valence-state (a3+CuA 2+.CuB + A32+) cytochrome oxidase with O2 at low temperature was studied by optical and e.p.r. spectroscopy. The existence of three intermediates [Clore & Chance (1978) Biochem. J. 173, 799-8101] was confirmed. From the e.p.r data it is clear that cytochrome a and CuA remain in the low-spin ferric and cupric states respectively throughout the reaction. No e.p.r. signals attributable to cytochrome a3 or CuB were seen in the intermediates. The difference spectra (intermediates minus unliganded mixed-valence-state cytochrome oxidase) and absolute spectra of the three intermediates were obtained. The chemcal nature of the three intermediates is discussed in terms of their spectroscopic properties. A catalytic cycle for cytochrome oxidase is proposed.

Chemical Phenomena↗
PubMed1978

The reaction of mercaptans with tyrosinases and hemocyanins.

1. Titration of Neurospora tyrosinase with 2-mercaptoethanol shows that the increase of absorbance at 700 nm is directly correlated to the loss of enzymatic activity. Approximately 2 mol of 2-mercaptoethanol per mole of protein are needed for full development of the green, enzymatically inactive complex. The increase of absorbance at 700 nm is also proportional to the intensity of the EPR signal and the amount of non-covalently bound 2-[35S] mercaptoethanol to the enzyme. The maximal EPR intensity reaches 70% of the protein concentration and at most 0.7--0.8 mol of 2-[35S] mercaptoethanol is bound per mol of enzyme. 2. Stopped-flow measurements show that in the reaction between 2-mercaptoethanol and Neurospora tyrosinase a raction intermediate with a strong absorption band at 360 nm is formed in an apparent second-order reaction. This intermediate displays no EPR-detectable signals. The intermediate decays in a similar complex fashion as the absorption band at 700 nm is formed. 3. The reaction of Neurospora tyrosinase with a variety of sulfhydryl compounds was also investigated. In most cases green coloured, enzymatically inactive complexes are formed displaying slightly different EPR signals. However, with cysteine and cysteamine violet coloured, enzymatically inactive complexes are formed which show rather different EPR signals. The integrated EPR intensities amount to 40--70% of the protein concentration. Based on simulations of 9 and 35 GHz spectra all observed EPR spectra can be represented as true S = 1/2 systems. The cysteamine complex can be interpreted as arising from a mixed valence Cu2+ . Cu+ complex. The 2-mercaptoethanol spectra can, however, arise from sulphur radicals. 4. Treatment of Agaricus bispora tyrosinase and Cancer pagures hemocyanin with 2-mercaptoethanol results in green-coloured, EPR detectable complexes similar to the one found with Neurospora tyrosinase. No such complexes are formed when hemocyanins from Helix pomatia and Panulirus interruptus were treated with this reagent.

Basidiomycota↗
PubMed1976

Low temperature magnetic susceptibility of a human Co(II) carbonic anhydrase B sulphonamide complex.

The magnetic susceptibility of the acetazolamide complex of human Co(II) carbonic anhydrase B was measured between 1.4 and 160 K. From the temperature dependence of the paramagnetic contribution of Co(II) a g-value of 2.23 +/- 0.02 and a zero-field splitting of (33 +/- 1) cm-1 were estimated. The effective number of Bohr magnetons as T leads to 0 is 3.35 in excellent agreement with the number (3.38) calculated from the apparent g'-values of the EPR spectrum at 16 K. Extrapolation to high temperatures gave an effective number of Bohr magnetons of 4.32.

Acetazolamide↗