Biomedical subjects

R Aasa

Publications and source records attributed to R Aasa.

At least 19 recordsLinked to original sources

PubMed2001

Chlorite dismutase from Ideonella dechloratans.

Chlorite dismutase has been purified from the chlorate-metabolizing bacterium Ideonella dechloratans. The purified enzyme is tetrameric, with a relative molecular mass of 25,000 for the subunit, and contains about 0.6 heme/subunit as isolated. Its catalytic properties are similar, but not identical, to those found for a similar enzyme purified earlier from the bacterium GR-1. The heme group in Ideonella chlorite dismutase is readily reduced by dithionite, in contrast to the GR-1 enzyme, and redox titration gave a value of -21 mV for the midpoint potential at pH 7. The heme group has been characterized by optical and EPR spectroscopy. It is high-spin ferric at neutral pH, with spectroscopic properties similar to those found for cytochrome c peroxidase. In the alkaline pH range, a low-spin compound is formed. A 22-residue N-terminal amino acid sequence has been determined and no homologue has been found in the protein sequence databases.

Amino Acid Sequence↗

PubMed2000

The distal heme center in Bacillus subtilis succinate:quinone reductase is crucial for electron transfer to menaquinone.

Succinate:quinone reductases are membrane-bound enzymes that catalyze electron transfer from succinate to quinone. Some enzymes in vivo reduce ubiquinone (exergonic reaction) whereas others reduce menaquinone (endergonic reaction). The succinate:menaquinone reductases all contain two heme groups in the membrane anchor of the enzyme: a proximal heme (heme b(P)) located close to the negative side of the membrane and a distal heme (heme b(D)) located close to the positive side of the membrane. Heme b(D) is a distinctive feature of the succinate:menaquinone reductases, but the role of this heme in electron transfer to quinone has not previously been analyzed. His28 and His113 are the axial ligands to heme b(D) in Bacillus subtilis succinate:menaquinone reductase. We have individually replaced these His residues with Leu and Met, respectively, resulting in assembled membrane-bound enzymes. The H28L mutant enzyme lacks succinate:quinone reductase activity probably due to a defective quinone binding site. The H113M mutant enzyme contains heme b(D) with raised midpoint potential and is impaired in electron transfer to menaquinone. Our combined experimental data show that the heme b(D) center, into which we include a quinone binding site, is crucial for succinate:menaquinone reductase activity. The results support a model in which menaquinone is reduced on the positive side of the membrane and the transmembrane electrochemical potential provides driving force for electron transfer from succinate via heme b(P) and heme b(D) to menaquinone.

Bacillus subtilis↗

PubMed1998

High-potential states of blue and purple copper proteins.

Electrochemical measurements show that there are high-potential states of two copper proteins, Pseudomonas aeruginosa azurin and Thermus thermophilus CuA domain; these perturbed states are formed in guanidine hydrochloride (GuHCl) solution in which the proteins are still blue (azurin) and purple (CuA). In each case, the high-potential state forms reversibly. Absorption (azurin, CuA), visible circular dichroism (azurin, CuA), resonance-Raman (CuA), and EPR (CuA) spectra indicate that the structure of the oxidized copper site of each high-potential form is very similar to that of the native protein. It is proposed that GuHCl perturbs one or more H-bonds in the blue or purple copper active site, thereby allowing Cu(I) to adopt a more favorable coordination structure than that in the rigid cavity of the native protein.

Azurin↗

PubMed1997

The type 2 copper of ascorbate oxidase.

Ascorbate oxidase, dissolved in Hepes or sodium phosphate buffers, was analyzed by EPR and activity measurements before and after storage at -30 degrees C and 77 K. The specific activity was somewhat higher in the phosphate buffer, about 3500-3700 Dawson units compared to about 3100 units of the enzyme dissolved in Hepes buffer. After storage at -30 degrees C the activity fell to 1400-2000 units in the phosphate buffer but only to 2600-2800 units in the Hepes buffer. Large changes occurred in the EPR spectrum of enzyme dissolved in the phosphate buffer after storing at -30 degrees C suggesting an alteration of the type 2 copper site. These changes were, however, reverted when the samples were thawed and rapidly frozen at 77 K. Copper analysis showed that about 50% of the total copper was EPR detected. The type 2 Cu2+ EPR intensity was in most samples close to 25% of the total EPR intensity. This low contribution of type 2 Cu2+ could not be changed if the enzyme was completely reduced and reoxidized, treated with Fe(CN)6(3), large excess of NaF, addition of 50% (v/v) ethylene glycol or dialyzed against 0.1 M Mes buffer (pH 5.5). Since the crystal structure shows that there are one each of types 1 and 2 copper in the monomers there must be another species with an EPR signal rather different from these two copper species. This signal is proposed to originate from some trinuclear centers. The EPR simulations show that it is possible to house a broad unresolved signal under the resolved type 1 and 2 signals so that the total integral becomes 50% of the total copper in the molecule.

Ascorbate Oxidase↗

PubMed1996

Site-directed mutagenesis of residues lining a putative proton transfer pathway in cytochrome c oxidase from Rhodobacter sphaeroides.

Several putative proton transfer pathways have been identified in the recent crystal structures of the cytochrome oxidases from Paracoccus denitrificans [Iwata et al. (1995) Nature 376, 660-669] and bovine [Tsukihara (1996) Science 272, 1138-1144]. A series of residues along one face of the amphiphilic transmembrane helix IV lie in one of these proton transfer pathways. The possible role of these residues in proton transfer was examined by site-directed mutagenesis. The three conserved residues of helix IV that have been implicated in the putative proton transfer pathway (Ser-201, Asn-207, and Thr-211) were individually changed to alanine. The mutants were purified, analyzed for steady-state turnover rate and proton pumping efficiency, and structurally probed with resonance Raman spectroscopy and FTIR difference spectroscopy. The mutation of Ser-201 to alanine decreased the enzyme turnover rate by half, and was therefore further characterized using EPR spectroscopy and rapid kinetic methods. The results demonstrate that none of these hydrophilic residues are essential for proton pumping or oxygen reduction activities, and suggest a model of redundant or flexible proton transfer pathways. Whereas previously reported mutants at the start of this putative channel (e.g., Asp-132-Asn) dramatically influence both enzyme turnover and coupling to proton pumping, the current work shows that this is not the case for all residues observed in this channel.

Carbon Monoxide↗

PubMed1996

Electron paramagnetic resonance studies of cobalt-substituted angiotensin I-converting enzyme.

Electron paramagnetic resonance (EPR) spectroscopy has been used to study the metal coordination sphere geometry in the cobalt-substituted Zn-protein angiotensin I-converting enzyme (ACE). It has been shown that ACE contains two distinct metal-binding sites. In the presence of the two structurally different inhibitors, captopril and ramiprilat, it is found that the metal binding sites are nearly structurally identical and are separated more than 10 A from each other. The metal atoms are most likely four- to five-coordinated, and it is argued that the inhibitor binds directly to the metal ion.

Animals↗

PubMed1996

Water-soluble, recombinant CuA-domain of the cytochrome ba3 subunit II from Thermus thermophilus.

Recently, the genes of cytochrome ba3 from thermus thermophilus [Keightley, J.A., et al. (1995) J. Biol. Chem. 270, 20345-20358], a homolog of the heme-copper oxidase family, have been cloned. We report here expression of a truncated gene, encoding the copper A (CuA) domain of cytochrome ba3, that is regulated by a T7 RNA polymerase promoter in Escherichia coli. The CuA-containing domain is purified in high yields as a water-soluble, thermostable, purple-colored protein. Copper analysis by chemical assay, mass spectrometry, X-ray fluorescence, and EPR spin quantification show that this protein contains two copper ions bound in a mixed-valence state, indicating that the CuA site in cytochrome ba3, is a binuclear center. The absorption spectrum of the CuA site, free of the heme interference in cytochrome ba3, is similar to the spectra of other soluble fragments from the aa3-type oxidase of Parachccus denitrificans [Lappalainen, P., et al. (1993) J. Biol Chem. 268, 26416-26421] and the caa3-type oxidase of Bacillus subtilis [von Wachenfeldt, C. et al. (1994) FEBS Lett. 340, 109-113]. There are intense bands at 480 nm (3100 M(-1) cm(-1)) and 530 nm (3200 M(-1) cm(-1)), a band in the near -IR centered at 790 nm (1900 M(-1) cn(-1)), and a weaker band at 363 nm (1300M(-1) cm(-1)). The visible CD spectrum shows a positive-going band at 460 nm and a negative-going band at 527 nm, the opposite signs of which may result from the binuclear nature of the site. The secondary structure prediction from the far-UV CD spectrum indicates that this domain is predominantly beta-sheet, in agreement with the recent X-ray structure reported for the complete P. denitrificans cytochrome aa3 molecule [Iwata, S., et al. (1995) Nature 376, 660-669] and the engineered, purple CyoA protein [Wilmanns, M., et al. (1996) Proc. Natl Acad. Sci. U.S.A. 92, 11955-11959]. However, the thermostability of the fragment described here (Tm approximately 80 degrees C) and the stable binding of copper over a broad pH range (pH 3-9) suggest this protein may be uniquely suitable for detailed physical-chemical study.

Amino Acid Sequence↗

PubMed1996

A ligand-exchange mechanism of proton pumping involving tyrosine-422 of subunit I of cytochrome oxidase is ruled out.

The molecular mechanism by which proton pumping is coupled to electron transfer in cytochrome c oxidase has not yet been determined. However, several models of this process have been proposed which are based on changes occurring in the vicinity of the redox centers of the enzyme. Recently, a model was described in which a well-conserved tyrosine residue in subunit I (Y422) was proposed to undergo ligand exchange with the histidine ligand (H419) of the high-spin heme a3 during the catalytic cycle, allowing both residues to serve as part of a proton transporting system. Site-directed mutants of Y422 have been constructed in the aa3-type cytochrome c oxidase of Rhodobacter sphaeroides to test this hypothesis (Y422A, Y422F). The results demonstrate that Y422 is not an essential residue in the electron transfer and proton pumping mechanisms of cytochrome c oxidase. However, the results support the predicted proximity of Y422 to heme a3, as now confirmed by crystal structure. In addition, it is shown that the pH-dependent reversed electron transfer between heme a and heme a3 is normal in the Y422F mutant. Hence, these data also demonstrate that Y422 is not the residue previously postulated to interact electrostatically with heme a3, nor is it responsible for the unique EPR characteristics of heme a in this bacterial oxidase.

Base Sequence↗

PubMed1996

Electron paramagnetic resonance studies of the soluble CuA protein from the cytochrome ba3 of Thermus thermophilus.

The electron paramagnetic resonance (EPR) spectrum of the binuclear CuA center in the water-soluble subunit II fragment from cytochrome ba3 of Thermus thermophilus was recorded at 3.93, 9.45, and 34.03 GHz, and the EPR parameters were determined by computer simulations. The frequency and M1 dependence of the linewidth was discussed in terms of g strain superimposed on a correlation between the A and g values. The g values were found to be gx = 1.996, gy = 2.011, gz = 2.187, and the two Cu ions contribute nearly equally to the hyperfine structure, with magnitude of Ax magnitude of approximately 15 G, magnitude of Ay magnitude = 29 G, and magnitude of Az magnitude of = 28.5 G (65Cu). Theoretical CNDO/S calculations, based on the x-ray structure of the Paracoccus denitrificans enzyme, yield a singly occupied antibonding orbital in which each Cu is pi*-bonded to one S and sigma*-bonded to the other. In contrast to the equal spin distribution suggested by the EPR simulations, the calculated contributions from the Cu ions differ by a factor of 2. However, only small changes in the ligand geometry are needed to reproduce the experimental results.

Bacterial Proteins↗

PubMed1995

EPR studies of wild-type and several mutants of cytochrome c oxidase from Rhodobacter sphaeroides: Glu286 is not a bridging ligand in the cytochrome a3-CuB center.

Wild-type and several mutants of cytochrome c oxidase from Rhodobacter sphaeroides were characterized by EPR spectroscopy. A pH-induced g12 signal, seen previously in mammalian cytochrome oxidase and assigned to the presence of a bridging carboxyl ligand in the bimetallic cytochrome a3-CuB site, is found also in the bacterial enzyme. Mutation of glutamate-286 to glutamine inactivates the enzyme but does not affect this signal, demonstrating that the carboxyl group of this residue is not the bridging ligand. Three mutants, M106Q, located one helix turn below a histidine ligand to cytochrome a, and T352A as well as F391Q, located close to the bimetallic center, are shown to affect dramatically the low-spin heme signal of cytochrome a. These mutants are essentially inactive, suggesting that these three mutations result in alterations to cytochrome a that render the oxidase non-functional.

Base Sequence↗

PubMed1995

Transmembrane topology and axial ligands to hemes in the cytochrome b subunit of Bacillus subtilis succinate:menaquinone reductase.

The membrane-anchoring subunit of Bacillus subtilis succinate:menaquinone reductase is a protein of 202 residues containing two protoheme IX groups with bis-histidine axial ligation. Residues His13, His28, His70, His113, and His155 are the possible heme ligands. The transmembrane topology of this cytochrome was analyzed using fusions to alkaline phosphatase. The results support a proposed model with five transmembrane polypeptide segments and the N-terminus exposed to the cytoplasm. Mutant B. subtilis cytochromes containing a His13-->Tyr, a His28-->Tyr, and a His113-->Tyr mutation, respectively, were produced in Escherichia coli, partially purified, and analyzed. In addition, succinate: menaquinone reductase containing the His13-->Tyr mutation in the anchor subunit was overproduced in B. subtilis, purified, and characterized. The data demonstrate that His13 is not an axial heme ligand. Thermodynamic and spectroscopic properties of the cytochrome are, however, affected by the His13-->Tyr mutation; compared to wild type, the redox potentials of both hemes are negatively shifted and the gmax signal in the EPR spectrum of the high-potential heme is shifted from 3.68 to 3.50. From the combined results we conclude that His28 and His113 function as axial ligands to the low-potential heme, which is located in the membrane near the outer surface of the cytoplasmic membrane. Residues His70 and His155 ligate the high-potential heme, which is positioned close to His13 in the protein, near the inner surface of the membrane.

Alkaline Phosphatase↗

PubMed1995

Multi-frequency EPR evidence for a binuclear CuA center in cytochrome c oxidase: studies with a 63Cu- and 65Cu-enriched, soluble domain of the cytochrome ba3 subunit II from Thermus thermophilus.

We have recorded multi-frequency EPR spectra of 63Cu- and 65Cu-labeled, water-soluble CuA-protein from the cytochrome ba3 of T. thermophilus. The spectrum taken at the highest frequency (34.03 GHz) shows no hyperfine structure and is nominally axial with apparent gz approximately 2.18 and gxy approximately 2.00. The spectrum taken at the lowest frequency (3.93 GHz) shows a rich hyperfine structure. Analyses of the spectra show that the observed splitting arises from an interaction of the unpaired electron with two Cu nuclei and support the notion that CuA is a mixed-valent [Cu(II)/Cu(I)] complex in which the unpaired electronic spin is distributed evenly over the two Cu ions.

Binding Sites↗

PubMed1993

Soluble CuA-binding domain from the Paracoccus cytochrome c oxidase.

In cytochrome c oxidase the C-terminal part of subunit II is outside the membrane and contains a copper center called CuA. We have expressed this domain of the Paracoccus denitrificans oxidase in a soluble form. Data obtained by quantitative copper-to-protein measurements, electrospray mass spectrometry, and electron paramagnetic resonance spectroscopy show that the center contains two copper atoms probably in a mixed valence configuration. Its absorbance spectrum is similar to that of the copper center A in nitrous oxide reductase. The EPR spectrum suggests that the center in the soluble protein is closely related to the native CuA site in the cytochrome oxidase complex. However, it seems likely that the copper center in the soluble domain is more exposed to the aqueous milieu than in the intact complex because its absorbance and EPR spectra are sensitive to pH. At alkaline pH one of the coppers in the site acquires type-2 character, indicating that it may be coordinated to a new ligand. The pK of this reversible change is about 8.2. The CuA-binding fragment is able to oxidize cytochrome c.

Amino Acid Sequence↗

PubMed1993

Spectral and physical properties of human extracellular superoxide dismutase: a comparison with CuZn superoxide dismutase.

Comparison of amino acid sequences have suggested similarities between the active site portions of the tetrameric, Cu- and Zn-containing glycoprotein, extracellular superoxide dismutase (EC-SOD) and the dimeric CuZn-SOD. In the present study spectral and physical properties of EC-SOD were analyzed to further knowledge about the enzyme and to extend the comparison with CuZn-SOD. EC-SOD displays an absorbance peak at 652 nm, blue-shifted some 30 nm compared with CuZn-SOD. The molar extinction coefficient, 167 M-1 cm-1 per copper atom, is similar to those of CuZn-SODs. In contrast to bovine and human CuZn-SOD, EC-SOD has a strong uv absorption originating from the rich content of aromatic amino acids. The extinction coefficient at 280 nm is 1.73 ml mg-1 cm-1. The electron paramagnetic resonance spectrum is also similar to those of bovine and human CuZn-SOD, but shows a slightly higher Az and a smaller difference between gx and gy. These spectral findings support the notion that the active sites of the SOD iso-enzymes are similar, but with a higher tetragonal symmetry of the copper ligands in EC-SOD. The circular dichroism spectrum of EC-SOD in the ultraviolet indicates a high alpha-helix content of the amino-terminal and carboxy-terminal parts flanking the central homologous active site portion. Similar to bovine CuZn-SOD, EC-SOD displays a marked physical resistance toward high temperature, pH extremes, and high urea and guanidinium chloride concentrations. This similarity in physical resistance suggests a central role of the homologous active site portion for the structural integrity of the SOD iso-enzymes.

Animals↗

PubMed1993

The nature of the CuA center in cytochrome c oxidase.

The merits of the suggestion that CuA in cytochrome oxidase is a mixed-valence binuclear site is reviewed on the basis of recent analytical and spectroscopic studies. First an alternative mononuclear model is presented. Metal analyses indicate that homogeneous oxidase preparations with high activity contain 3Cu/2Fe. Multifrequency EPR measurements demonstrate a close similarity with a copper site in nitrous oxide reductase, and this is also supported by optical and MCD spectra. Strong evidence for a binuclear site is provided by a 7-line hyperfine structure in the EPR spectra of both enzymes. A binuclear model consistent with amino acid sequence data can be formulated.

Amino Acid Sequence↗

PubMed1992

Two hemes in Bacillus subtilis succinate:menaquinone oxidoreductase (complex II).

Succinate:menaquinone-7 oxidoreductase (complex II) of the Gram-positive bacterium Bacillus subtilis consists of equimolar amounts of three polypeptides; a 65-kDa FAD-containing polypeptide, a 28-kDa iron-sulfur cluster containing polypeptide, and a 23-kDa membrane-spanning cytochrome b558 polypeptide. The enzyme complex was overproduced 2-3-fold in membranes of B. subtilis cells containing the sdhCAB operon on a low copy number plasmid and was purified in the presence of detergent. The cytochrome b558 subunit alone was similarly overexpressed in a complex II deficient mutant and partially purified. Isolated complex II catalyzed the reduction of various quinones and also quinol oxidation. Both activities were efficiently albeit not completely blocked by 2-n-heptyl-4-hydroxyquinoline N-oxide. Chemical analysis demonstrated two protoheme IX per complex II. One heme component was found to have an Em,7.4 of +65 mV and an EPR gmax signal at 3.68, to be fully reducible by succinate, and showed a symmetrical alpha-band absorption peak at 555 nm at 77 K. The other heme component was found to have an Em,7.4 of -95 mV and an EPR gmax signal at 3.42, was not reducible by succinate under steady-state conditions, and showed in the reduced state an apparent split alpha-band absorption peak with maxima at 553 and 558 nm at 77 K. Potentiometric titrations of partially purified cytochrome b558 subunit demonstrated that the isolated cytochrome b558 also contains two hemes. Some of the properties, i.e., the alpha-band light absorption peak at 77 K, the line shapes of the EPR gmax signals, and reactivity with carbon monoxide were observed to be different in B. subtilis cytochrome b558 isolated and in complex II. This suggests that the bound flavoprotein and iron-sulfur protein subunits protect or affect the heme environment in the assembled complex.

Bacillus subtilis↗

PubMed1992

Multifrequency EPR investigations into the origin of the S2-state signal at g = 4 of the O2-evolving complex.

The low-temperature S2-state EPR signal at g = 4 from the oxygen-evolving complex (OEC) of spinach Photosystem-II-enriched membranes is examined at three frequencies, 4 GHz (S-band), 9 GHz (X-band) and 16 GHz (P-band). While no hyperfine structure is observed at 4 GHz, the signal shows little narrowing and may mask underlying hyperfine structure. At 16 GHz, the signal shows g-anisotropy and a shift in g-components. The middle Kramers doublet of a near rhombic S = 5/2 system is found to be the only possible origin consistent with the frequency dependence of the signal. Computer simulations incorporating underlying hyperfine structure from an Mn monomer or dimer are employed to characterize the system. The low zero field splitting (ZFS) of D = 0.43 cm-1 and near rhombicity of E/D = 0.25 lead to the observed X-band g value of 4.1. Treatment with F- or NH3, which compete with Cl- for a binding site, increases the ZFS and rhombicity of the signal. These results indicate that the origin of the OEC signal at g = 4 is either an Mn(II) monomer or a coupled Mn multimer. The likelihood of a multimer is favored over that of a monomer.

Electron Spin Resonance Spectroscopy↗

PubMed1992

Restoration of a lost metal-binding site: construction of two different copper sites into a subunit of the E. coli cytochrome o quinol oxidase complex.

The cupredoxin fold, a Greek key beta-barrel, is a common structural motif in a family of small blue copper proteins and a subdomain in many multicopper oxidases. Here we show that a cupredoxin domain is present in subunit II of cytochrome c and quinol oxidase complexes. In the former complex this subunit is thought to bind a copper centre called CuA which is missing from the latter complex. We have expressed the C-terminal fragment of the membrane-bound CyoA subunit of the Escherichia coli cytochrome o quinol oxidase as a water-soluble protein. Two mutants have been designed into the CyoA fragment. The optical spectrum shows that one mutant is similar to blue copper proteins. The second mutant has an optical spectrum and redox potential like the purple copper site in nitrous oxide reductase (N2OR). This site is closely related to CuA, which is the copper centre typical of cytochrome c oxidase. The electron paramagnetic resonance (EPR) spectra of both this mutant and the entire cytochrome o complex, into which the CuA site has been introduced, are similar to the EPR spectra of the native CuA site in cytochrome oxidase. These results give the first experimental evidence that CuA is bound to the subunit II of cytochrome c oxidase and open a new way to study this peculiar copper site.

Amino Acid Sequence↗