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M Wikström

Publications and source records attributed to M Wikström.

At least 37 records · Page 2Linked to original sources
PubMed2000

The role of the D- and K-pathways of proton transfer in the function of the haem-copper oxidases.

The X-ray structures of several haem-copper oxidases now at hand have given important constraints on how these enzymes function. Yet, dynamic data are required to elucidate the mechanisms of electron and proton transfer, the activation of O(2) and its reduction to water, as well as the still enigmatic mechanism by which these enzymes couple the redox reaction to proton translocation. Here, some recent observations will be briefly reviewed with special emphasis on the functioning of the so-called D- and K-pathways of proton transfer. It turns out that only one of the eight protons taken up by the enzyme during its catalytic cycle is transferred via the K-pathway. The D-pathway is probably responsible for the transfer of all other protons, including the four that are pumped across the membrane. The unique K-pathway proton may be specifically required to aid O-O bond scission by the haem-copper oxidases.

Binding Sites↗
PubMed2000

Proton translocation by cytochrome c oxidase can take place without the conserved glutamic acid in subunit I.

A glutamic acid residue in subunit I of the heme-copper oxidases is highly conserved and has been directly implicated in the O(2) reduction and proton-pumping mechanisms of these respiratory enzymes. Its mutation to residues other than aspartic acid dramatically inhibits activity, and proton translocation is lost. However, this glutamic acid is replaced by a nonacidic residue in some structurally distant members of the heme-copper oxidases, which have a tyrosine residue in the vicinity. Here, using cytochrome c oxidase from Paracoccus denitrificans, we show that replacement of the glutamic acid and a conserved glycine nearby lowers the catalytic activity to <0.1% of the wild-type value. But if, in addition, a phenylalanine that lies close in the structure is changed to tyrosine, the activity rises more than 100-fold and proton translocation is restored. Molecular dynamics simulations suggest that the tyrosine can support a transient array of water molecules that may be essential for proton transfer in the heme-copper oxidases. Surprisingly, the glutamic acid is thus not indispensable, which puts important constraints on the catalytic mechanism of these enzymes.

Amino Acid Sequence↗
PubMed2000

O-O bond splitting mechanism in cytochrome oxidase.

Hybrid density functional theory (DFT) calculations have been used to investigate different mechanisms for O-O bond splitting in cytochrome oxidase. It is shown that the requirement for a low activation barrier for the O-O bond splitting is that two protons, apart from the tyrosine hydroxyl proton, are available at the binuclear center. A mechanism is suggested for the transformation from a species with a molecularly coordinated O2, to an O-O cleaved species with an oxo-ferryl group. The mechanism has a calculated activation barrier in reasonable agreement with experimental estimates, and the overall reaction is close to thermoneutral, in line with the requirement that the energy wasted as heat should be minimized. The rate limiting step in the mechanism occurs at the initial Fe-O2 intermediate, consistent with experimental observations that the decay of the oxy intermediate parallels the increase of the oxo product. The formation of a radical at the cross-linked tyrosine-histidine structure is a possible source for one of the electrons required in the bond cleavage process. Possible sources for the two protons are discussed, including a suggested key role for the hydroxyl group on the farnesyl side chain of heme a3.

Animals↗
PubMed2000

Binding of O(2) and its reduction are both retarded by replacement of valine 279 by isoleucine in cytochrome c oxidase from Paracoccus denitrificans.

The crystal structure of the heme-copper oxidases suggested a putative channel of oxygen entry into the heme-copper site of O(2) reduction. Changing a conserved valine near this center in cytochrome bo(3) of Escherichia coli to isoleucine caused a significant increase in the apparent K(M) for oxygen with little or no change in V(max), suggesting that oxygen diffusion had been partially blocked [Riistama, S., Puustinen, A., García-Horsman, A., Iwata, S., Michel, H., and Wikström, M. (1996) Biochim. Biophys. Acta 1275, 1-4]. To study this phenotype further using rapid kinetic methods, the corresponding change (V279I) has been made in cytochrome aa(3) from Paracoccus denitrificans. In this mutant, the apparent K(M) for oxygen is 8 times higher than in the wild-type enzyme, whereas V(max) is decreased only to approximately half of the wild-type value. Flow-flash kinetic measurements show that the initial binding of oxygen to the heme of the binuclear site is indeed much slower in the mutant than in the wild-type enzyme. However, the subsequent phases of the reaction with O(2) are also slow although the pure heme-to-heme electron transfer process is essentially unperturbed. It is suggested that the mutation sterically hinders O(2) entry into the binuclear site and that it may also perturb the structure of local water molecules involved in proton transfer to this site.

Amino Acid Substitution↗
PubMed2000

Proton translocation by cytochrome c oxidase: a rejoinder to recent criticism.

Ten years ago, intermediate reaction steps in the catalytic cycle of cytochrome c oxidase were titrated with phosphorylation potential in isolated mitochondria, and the results were interpreted as evidence for thermodynamic linkage of proton translocation exclusively to the oxidative reaction steps of the catalytic cycle [Wikström, M. (1989) Nature 338, 776-778]. Michel has recently argued that this work was flawed, and proposed a mechanism in which one of the four steps of proton translocation is linked to the reductive phase of the catalytic cycle [Michel, H. (1999) Biochemistry 38, 15129-15140]. Here, the original data are scrutinized and related to information that has accumulated since this work was published. The analysis shows that the main conclusions from this work still hold. Michel's mechanism of proton translocation is briefly discussed, and found to be at odds with some experimental observations.

Electron Transport↗
PubMed2000

Solution structure and dynamics of the DNA-binding domain of the adipocyte-transcription factor FREAC-11.

Transcription factors of the forkhead type share a highly conserved DNA-binding domain of about 100 amino acid residues. FREAC-11, expressed in adipocytes, belongs to this class. Here, we report on NMR studies that established the three-dimensional structure of the FREAC-11, DNA-binding domain. Although apparent similarities to the structures of other members within the forkhead family are observed, the structure also reveals some remarkable differences. Along with the complementary dynamics, the data provide insight into the fundamentals of sequence specificity within a highly conserved motif.

Adipose Tissue↗
PubMed2000

Interaction between the formyl group of heme a and arginine 54 in cytochrome aa(3) from Paracoccus denitrificans.

The optical spectrum of heme a is red-shifted in aa(3)-type cytochrome c oxidases compared to isolated low-spin heme A model compounds. Early spectroscopic studies indicated that this may be due to hydrogen-bonding of the formyl group of heme a to an amino acid in the close vicinity. Here we show that most of the optical spectral shift of native heme a is due to a hydrogen-bonding interaction between the formyl group and arginine-54 in subunit I of cytochrome aa(3) from Paracoccus denitrificans, and that a smaller part is due to an electrostatic interaction between the D ring propionate of heme a and arginine-474.

Arginine↗
PubMed2000

The structure of the ubiquinol oxidase from Escherichia coli and its ubiquinone binding site.

Cell respiration is catalyzed by the heme-copper oxidase superfamily of enzymes, which comprises cytochrome c and ubiquinol oxidases. These membrane proteins utilize different electron donors through dissimilar access mechanisms. We report here the first structure of a ubiquinol oxidase, cytochrome bo3, from Escherichia coli. The overall structure of the enzyme is similar to those of cytochrome c oxidases; however, the membrane-spanning region of subunit I contains a cluster of polar residues exposed to the interior of the lipid bilayer that is not present in the cytochrome c oxidase. Mutagenesis studies on these residues strongly suggest that this region forms a quinone binding site. A sequence comparison of this region with known quinone binding sites in other membrane proteins shows remarkable similarities. In light of these findings we suggest specific roles for these polar residues in electron and proton transfer in ubiquinol oxidase.

Amino Acid Sequence↗
PubMed2000

Reliability and validity of a Swedish version of the Revised Leeds Disability Questionnaire for patients with ankylosing spondylitis.

The aim of the study was to investigate the reliability and the validity of a Swedish version of the Revised Leeds Disability Questionnaire (RLDQ). Forty-two patients, 36 men and 6 women, median age 52.5 years, median symptom duration 24 years, with spondylarthropathy were assessed with the RLDQ, three questions on the content of the questionnaire, and range-of-motion measures. The results indicated satisfactory test-retest stability and internal consistency, and only minor internal redundancy. RLDQ items were generally considered relevant and suggested additions were similar to items already included in the questionnaire. Patients with low disability scores tended to be older and have better joint mobility than those with higher scores. Physiotherapists observed and scored disability significantly higher than patients. Correlations between subscores of the RLDQ and range-of-motion measures were in the main weak. In conclusion the Swedish version of the RLDQ may be considered as reliable and valid.

Adult↗
PubMed1999

Glutamate-89 in subunit II of cytochrome bo3 from Escherichia coli is required for the function of the heme-copper oxidase.

Recent electrostatics calculations on the cytochrome c oxidase from Paracoccus denitrificans revealed an unexpected coupling between the redox state of the heme-copper center and the state of protonation of a glutamic acid (E78II) that is 25 A away in subunit II of the oxidase. Examination of more than 300 sequences of the homologous subunit in other heme-copper oxidases shows that this residue is virtually totally conserved and is in a cluster of very highly conserved residues at the "negative" end (bacterial cytoplasm or mitochondrial matrix) of the second transmembrane helix. The functional importance of several residues in this cluster (E89II, W93II, T94II, and P96II) was examined by site-directed mutagenesis of the corresponding region of the cytochrome bo(3) quinol oxidase from Escherichia coli (where E89II is the equivalent of residue E78II of the P. denitrificans oxidase). Substitution of E89II with either alanine or glutamine resulted in reducing the rate of turnover to about 43 or 10% of the wild-type value, respectively, whereas E89D has only about 60% of the activity of the control oxidase. The quinol oxidase activity of the W93V mutant is also reduced to about 30% of that of the wild-type oxidase. Spectroscopic studies with the purified E89A and E89Q mutants indicate no perturbation of the heme-copper center. The data suggest that E89II (E. coli numbering) is critical for the function of the heme copper oxidases. The proximity to K362 suggests that this glutamic acid residue may regulate proton entry or transit through the K-channel. This hypothesis is supported by the finding that the degree of oxidation of the low-spin heme b is greater in the steady state using hydrogen peroxide as an oxidant in place of dioxygen for the E89Q mutant. Thus, it appears that the inhibition resulting from the E89II mutation is due to a block in the reduction of the heme-copper binuclear center, expected for K-channel mutants.

Alanine↗
PubMed1999

The calcium binding site in cytochrome aa3 from Paracoccus denitrificans.

A shift in the spectrum of heme a induced by calcium or proton binding, or by the proton electrochemical gradient, has been attributed to interaction of Ca2+ or H+ with the vicinity of the heme propionates in mitochondrial cytochrome c oxidase, and proposed to be associated with the exit path of proton translocation. However, this shift is absent in cytochrome c oxidases from yeast and bacteria [Kirichenko et al. (1998) FEBS Lett. 423, 329-333]. Here we report that mutations of Glu56 or Gln63 in a newly described Ca2+/Na+ binding site in subunit I of cytochrome c oxidase from Paracoccus denitrificans [Ostermeier et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 10547-10553] establish the Ca2+-dependent spectral shift in heme a. This shift is counteracted by low pH and by sodium ions, as was described for mammalian cytochrome c oxidase, but in the mutant Paracoccus enzymes Na+ is also able to shift the heme a spectrum, albeit to a smaller extent. We conclude that the Ca2+-induced shift in both Paracoccus and mitochondrial cytochrome aa3 is due to binding of the cation to the new metal binding site. Comparison of the structures of this site in the two types of enzyme allows rationalization of their different reactivity with cations. Structural analysis and data from site-directed mutagenesis experiments suggest mechanisms by which the cation binding may influence the heme spectrum.

Amino Acid Sequence↗
PubMed1999

Proton translocation by cytochrome c oxidase.

Cell respiration in mitochondria and some bacteria is catalysed by cytochrome c oxidase, which reduces O2 to water, coupled with translocation of four protons across the mitochondrial or bacterial membrane. The enzyme's catalytic cycle consists of a reductive phase, in which the oxidized enzyme receives electrons from cytochrome c, and an oxidative phase, in which the reduced enzyme is oxidized by O2. Previous studies indicated that proton translocation is coupled energetically only to the oxidative phase, but this has been challenged. Here, with the purified enzyme inlaid in liposomes, we report time-resolved measurements of membrane potential, which show that half of the electrical charges due to proton-pumping actually cross the membrane during reduction after a preceding oxidative phase. pH measurements confirm that proton translocation also occurs during reduction, but only when immediately preceded by an oxidative phase. We conclude that all the energy for proton translocation is conserved in the enzyme during its oxidation by O2. One half of it is utilized for proton-pumping during oxidation, but the other half is unlatched for this purpose only during re-reduction of the enzyme.

Animals↗
PubMed1999

Coordination of CuB in reduced and CO-liganded states of cytochrome bo3 from Escherichia coli. Is chloride ion a cofactor?

The ubiquinol oxidase cytochrome bo3 from Escherichia coli is one of the respiratory heme-copper oxidases which catalyze the reduction of O2 to water linked to translocation of protons across the bacterial or mitochondrial membrane. We have studied the structure of the CuB site in the binuclear heme-copper center of O2 reduction by EXAFS spectroscopy in the fully reduced state of this enzyme, as well as in the reduced CO-liganded states where CO is bound either to the heme iron or to CuB. We find that, in the reduced enzyme, CuB is coordinated by one weakly bound and two strongly bound histidine imidazoles at Cu-N distances of 2.10 and 1.92 A, respectively, and that an additional feature at 2.54 A is due to a highly ordered water molecule that might be weakly associated with the copper. Unexpectedly, the binding of CO to heme iron is found to result in a major conformational change at CuB, which now binds only two equidistant histidine imidazoles at 1.95 A and a chloride ion at 2. 25 A, with elimination of the water molecule and one of the histidines. Attempts to remove the chloride from the enzyme by extensive dialysis did not change this finding, nor did substitution of chloride with bromide. Photolysis of CO bound to the heme iron is known to cause the CO to bind to CuB in a very fast reaction and to remain bound to CuB at low temperatures. In this state, we indeed find the CO to be bound to CuB at a Cu-C distance of 1.85 A, with chloride still bound at 2.25 A and the two histidine imidazoles at a Cu-N distance of 2.01 A. These results suggest that reduction of the binuclear site weakens the bond between CuB and one of its three histidine imidazole ligands, and that binding of CO to the reduced binuclear site causes a major structural change in CuB in which one histidine ligand is lost and replaced by a chloride ion. Whether chloride is a cofactor in this enzyme is discussed.

Animals↗
PubMed1999

Assignment and charge translocation stoichiometries of the major electrogenic phases in the reaction of cytochrome c oxidase with dioxygen.

The reaction of cytochrome c oxidase with dioxygen has been studied by means of time-resolved measurements of electrical membrane potential (DeltaPsi). Microsecond time resolution was achieved by starting with the CO-inhibited enzyme, which was photolyzed after addition of oxygen. The time course of the reaction could be fitted by using a five-step sequential reaction as a model. The first two phases of the reaction, which correspond in time to binding of oxygen followed by formation of the P (peroxy) intermediate, as observed spectroscopically, are not associated with net charge displacement across the membrane. After this lag, DeltaPsi develops in three phases, which correspond in time to the conversion of P to the F (ferryl) intermediate, in a single phase, and conversion of F to O (the fully oxidized enzyme), in two phases. The amplitude of DeltaPsi was approximately equal for the P --> F and F --> O portions of the reaction. When the oxygen reaction is started with incompletely reduced enzyme, it will halt at the P or F state. When the reaction was allowed to proceed to the F state, but no further, only the fast phase of DeltaPsi formation was observed, whereas no DeltaPsi was generated if the reaction was halted at P. This finding places the assignments of phases in the electrometric data on a firmer basis-they are no longer based solely on temporal correspondence with phases in the spectroscopic data. To define the number of charges transferred across the membrane during the reaction, some kind of calibration is needed. For this purpose, another type of reaction-electron transfer following CO photolysis in the absence of oxygen ("backflow")-was studied. Parallel spectroscopic and electrometric measurements showed that the fast electron transfer from the low-spin heme to CuA in the backflow process results in approximately 11 times smaller amplitude of DeltaPsi as compared with DeltaPsi generated in the reaction of the reduced enzyme with oxygen (the polarity is also reversed). If it is assumed that transfer of an electron from the low-spin heme to CuA amounts to movement of a unit charge across half of the membrane dielectric, charge translocation in the reaction of the reduced enzyme with oxygen amounts to approximately 5.5 unit charges-the value predicted if all four protons pumped during the catalytic cycle are translocated during the oxidative part of the reaction.

Animals↗
PubMed1999

Proton exit from the heme-copper oxidase of Escherichia coli.

Pathways of proton entry have been identified in the proton-translocating heme-copper oxidases, but the proton exit pathway is unknown. Here we report experiments with cytochrome bo3 in Escherichia coli cells that may identify the beginning of the exit pathway. Systematic mutations of arginines 438 and 439 (R481 and R482 in the E. coli enzyme), numbering as in cytochrome aa3 from bovine heart mitochondria, which interact with the ring D propionates of the two heme groups, reveal that the D propionate of the oxygen-binding heme is involved in proton pumping; its anionic form must be stabilized in order for proton translocation to occur. This may locate the beginning of the pathway by which pumped protons exit from the enzyme structure.

Biological Transport↗