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H Taniuchi

Publications and source records attributed to H Taniuchi.

At least 37 records · Page 2Linked to original sources
PubMed1987

Intramolecular flip between two alternative forms of complex formed from a heme fragment and apoprotein of horse cytochrome c.

The previous studies have shown that (a) noncovalent interactions of the ferro-heme fragment of residues 1-38 and apoprotein (1-104) of horse cytochrome c simultaneously and specifically form two isomeric complexes, types I and II resembling the native protein (the redundant residues flexibly protruding from the ordered structure); (b) the type II form but not type I appears to bind to CO; and (c) residues 39-55 are more flexible for type II form than type I (Parr, G. R., and Taniuchi, H. (1981) J. Biol. Chem. 256, 125-132). In the present study, we investigated 1) kinetics and thermodynamics of interconversion between type I and II forms of complex ferro-(1-38)-H.(1-104); 2) the properties of the CO binding population; 3) the rate of dissociation of complexes ferri- and ferro-(1-38)-H.(39-104) (mimicking type II form); and 4) thermal transition of the 695-nm absorption band and biological activity of complexes. The results indicate (a) interconversion between type I and II forms of complex ferro-(1-38)-H.(1-104) occurs without going through dissociation (t1/2 less than or equal to 12 min at 10 degrees C) and is associated with delta H (= -7.2 +/- 3.7 kcal/mol at 10 degrees C) favoring type I form and delta S (= 23 +/- 13 e.u. at 10 degrees C) favoring type II; (b) the CO-binding population correlates with type II; and (c) change from the ferrous to the ferric state of heme appears to perturb the thermodynamic relationship between type I and II forms. Interpreting the results and available evidence, we suggest that "intramolecular" flip between ferro-type I and ferro-type II forms would establish the Boltzmann distribution of these two distinctly different energy states, type I form having more strengthened interatomic interactions and type II more pronounced internal motion.

Animals↗
PubMed1986

A study of roles of evolutionarily invariant proline 30 and glycine 34 of cytochrome c.

The previous studies (Juillerat, M. A., and Taniuchi, H. (1986) J. Biol. Chem. 261, 2697-2711), using a three-fragment complex (1-25)H X (28-38) X (39-104) of horse cytochrome c, have shown that invariant leucine 32 and partially invariant leucine 35, both buried in the interior, exhibit a striking difference in perturbation of binding fragment (28-38) by substitution with isoleucine. Then the idea has been proposed that the energy states of leucine 32, the Met-80-S-heme-Fe bond and other distant residues such as tryptophan 59 would be coupled to generate extra force while leucine 35 would be less important for such coupling if it were involved. In the present studies we synthesized three (28-38) analogs substituting invariant proline 30 with glycine or invariant glycine 34 with alanine or serine. Thermodynamic and kinetic studies and UV CD and biological activity measurements were carried out on binding of the analogs to complex (1-25)H X (39-104). The results with the ferric form show that perturbations of delta G, delta H, and delta S associated with formation of the intermediate complex and with the ensuing process by the Gly34----Ala or Ser substitution result in weakening the Met-80-S-heme-Fe bond formed in the latter process; in contrast, perturbation by the Pro30----Gly substitution is small. However, the biological activity is more perturbed by the Pro30----Gly substitution than by the Gly34----Ala or Ser; and in the Gly34----Ala or Ser substitution the complex appears to be more readily activated for both formation and disruption of the Met-80-S-heme-Fe bond at 20 degrees C and below than without substitution. In all cases reduction of the heme strengthens the binding of fragment (28-38). However, striking are the increases in perturbation (less negative) of both delta H and delta S for binding of fragment (28-38) to form the ground state on reduction of the heme in the Pro30----Gly, Gly34----Ala or Ser (the present studies), and Leu32----norvaline (the previous studies) substitutions. It is known that fluctuation of the atomic positions of most residues of tuna ferrocytochrome c including Pro30, Leu32, and Gly34 increases on oxidation of the heme and that these three residues are among those showing the least fluctuating atomic positions (Takano, T., and Dickerson, R.E. (1982) in Electron Transport and Oxygen Utilization (Ho, C., ed) pp. 17-26, Elsevier/North-Holland Biomedical Press, New York).(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1986

A study on the role of evolutionarily invariant leucine 32 of cytochrome c.

To investigate the role of evolutionarily invariant leucine 32 of horse cytochrome c, analogs of residues 28-38, (28-38), each containing a substituted amino acid at positions 32 or 35 were synthesized using Merrifield's method. Position 35 is leucine in horse cytochrome c but replaced by nonpolar amino acids in some species. The ability of the analogs to bind to the two-fragment complex of ferri- or ferro heme fragment (1-25)H and apofragment (39-104) was measured using gel filtration and equilibrium dialysis. Replacement of leucine 32 with isoleucine, for example, increased the dissociation constant by more than 400-fold for the ferrous complex. In contrast, replacement of leucine 35 with isoleucine seems to increase it only by a small degree. Since both leucine 32 and leucine 35 are completely buried within the structure, hydrophobic interaction would not explain this striking difference. However, thermodynamic analyses and absorption spectra of the ferric complex have indicated that replacement with norvaline of leucine 32 increases both delta H and delta S (more positive) associated with formation of an intermediate three-fragment complex and decreases both delta H and delta S (more negative) associated with transformation from the intermediate to the ground state, resulting in weakening the methionine 80--S--heme-Fe bond formed in the latter step. Taking the results together with the fragment exchange studies on the ferrous complex and available evidence, we suggest that the interaction involving leucine 32 would be coupled not only with the methionine 80--S--heme-Fe bond but also with the energy state of other distant residues such as tryptophan 59, generating extra energy for modulating the binding of the complex, i.e. the force of folding. In contrast, leucine 35 would be less important even if it were involved in such coupling.

Amino Acid Sequence↗
PubMed1986

Extremely fast hydrogen exchange of ribonuclease-(1-118) as compared with native RNase A and its implication for the conformational energy state.

RNase-(1-118) containing native disulfide bonds is similar in fold to native RNase A but not of lowest Gibbs energy as compared with the isomers containing non-native disulfide bonds. The present n.m.r. studies have indicated a dramatic increase in the exchange rate of all of the 'protected' amide protons of RNase-(1-118) over RNase A. A calculation shows a large increase in the rate of 'opening' of the structure. The exchange rate of the protected amide protons of RNase-(1-120) is slower than RNase-(1-118) but much faster than RNase A. Binding with a synthetic complementing fragment (114-124) markedly reduces the exchange rate of 20 to 25 amide protons of RNase-(1-118). It has previously been shown that binding with a complementing fragment of RNase-(1-118) generates a lowest Gibbs energy state. Thus, using available thermodynamic information for interpretation, we suggest that a) removal of six carboxy terminal residues of RNase A would disrupt coupling between these residues and those distant in the structure (loss of extra stabilizing energy), b) this would, in turn, alter the enthalpy-entropy compensation in such a way that the magnitude of Gibbs energy change favoring folding is significantly reduced without a large change of fold and c) in this activated state the molecule would be highly motile.

Disulfides↗
PubMed1985

On the specificity of cytochrome c synthetase in recognition of the amino acid sequence of apocytochrome c.

Two forms of yeast cytochrome c synthetases with different specificities were resolved, one (synthetase I), solubilized from mitochondria or the cell debris with Triton X-100, recognizing not horse apocytochrome c but yeast apo-iso-1-cytochrome c as a substrate and the other (synthetase II) still bound with the particulate fraction from mitochondria after treatment with Triton, recognizing both horse and yeast apocytochromes c. The activity with labeled yeast apo-iso-1-cytochrome c as a substrate of cytochrome c synthetase I can be quantitatively inhibited by nonlabeled Candida krusei apocytochrome c and partially by nonlabeled tuna apocytochrome c but not by nonlabeled horse apocytochrome c indicating a specific amino acid sequence being recognized. However, an enzyme similarly solubilized from beef heart mitochondria recognized both horse apocytochrome c and yeast apo-iso-1-cytochrome c for attachment of heme. In view of the fact that the yeast synthetase II and the beef synthetase can both utilize either horse apocytochrome c or yeast apo-iso-1-cytochrome c as substrates, we suggest that these enzymes may also be involved in biosynthesis of cytochrome c1, that is, the ability to attach heme to apocytochrome c and apocytochrome c1 may have been conserved in eucaryotic cells, and that both synthetases may therefore be homologous.

Amino Acid Sequence↗
PubMed1984

Synthesis of a heme fragment of horse cytochrome c which forms a productive complex with a native apofragment.

To investigate the amino acid sequence of apocytochrome c recognized by yeast mitochondrial cytochrome c synthetase, a labeled apofragment containing residues 1 to 25 of horse cytochrome c, N alpha-[3H]acetyl-(1-25), and an analog containing glycine in place of cysteines 14 and 17, N alpha-[3H]acetyl-[14-Gly, 17-Gly] (1-25), have been synthesized using the Merrifield's improved solid phase method (Mitchell, A. R., Ericksen , B. W., Ryabtsev , M. N., Hodges , R. S., and Merrifield, R. B. (1976) J. Am. Chem. Soc. 98, 7357-7362) and then purified to homogeneity. Upon incubation with yeast mitochondria in the presence of hemin, a radioactive species, produced from N alpha-[3H]acetyl-(1-25) and not from N alpha-acetyl-[14-Gly, 17-Gly] (1-25), formed a complex with native apofragment (23-104). This semisynthetic complex was indistinguishable from the native complex in resistance to trypsin upon reduction with ascorbate and by ion-exchange chromatography. The radioactive species, dissociated from the complex was identical with native heme fragment N alpha-acetyl-(1-25)H by reverse-phase high pressure liquid chromatography. Treatment of this radioactive heme fragment with silver sulfate and then with dithiothreitol generated the original apofragment . Thus, if it is assumed that the mitochondrial enzyme catalyzing this covalent attachment of heme to synthetic apo-N alpha-[3H]acetyl-(1-25) is a cytochrome c synthetase, the results may be interpreted as indicating that the amino acid sequence of residues 1 to 25 of horse cytochrome c would contain the principal recognition site of the enzyme.

Amino Acid Sequence↗
PubMed1983

Evidence for formation of two thioether bonds to link heme to apocytochrome c by partially purified cytochrome c synthetase.

Cytochrome c synthetase has been solubilized from yeast mitochondria using Triton X-100 and fractionated with ammonium sulfate. Use of this partially purified enzyme has permitted us to isolate a quantity of iso-1-cytochrome c formed from 125I-labeled apocytochrome c and hemin in the presence of a NADPH-generating system. Visible absorption spectra (pH 8.0 or 5.0) including alpha, beta, and Soret bands and their molar absorption coefficients of this enzymatically synthesized cytochrome c in the oxidized and reduced states are the same, within experimental error, as those of native cytochrome c. Pyridine ferrohemochrome (pH 13) of the synthesized species also exhibits the same alpha and beta bands as those of iso-l-cytochrome c and similar to those reported for heme peptides of cytochrome c. If only one or no thioether bond were formed between the two vinyl side groups of heme and the cysteine residues of apocytochrome c, all these alpha and beta bands would have shifted to red (Pettigrew, G. W., Leaver, J. L., Meyer, T. E., and Ryle, T. E. (1975) Biochem J. 147, 291-302). Thus, two thioether bonds appear to be formed to link heme to apocytochrome c by cytochrome c synthetase, completing information of the three-dimensional structure of cytochrome c.

Cytochrome c Group↗
PubMed1983

Evidence of a compact structure for kinetic intermediates in the folding of a fragment complex of tuna cytochrome c.

A noncovalently bound, ordered complex consisting of fragments (1-25)H and (14-103) derived from tuna cytochrome c has been prepared and characterized. The equilibrium properties of this complex are indistinguishable from those of the corresponding complex, (1-25)H X (23-104), of horse cytochrome c. The tuna species possesses a second tryptophan residue (at position 33 of the amino acid sequence and replacing the histidine residue of the horse protein) whose fluorescence is also essentially fully quenched in the native form and in the complex. It is found that for the tuna complex, the fluorescence of both tryptophans is fully quenched during the second order phase of the reaction mechanism. This provides evidence for the compact nature of the intermediate complexes formed at this stage of the complementation process (Parr, G. R., and Taniuchi, H. (1982) J. Biol. Chem. 257, 10103-10111). The observed second order rate constant for the tuna fragments is reduced by a factor of two from that for the horse fragments. During the first order kinetic phase corresponding to the transition from the intermediate complexes to the native complex, qualitative as well as quantitative differences are also observed between the tuna and the horse species. The kinetics of complementation of the "hybrid" complexes is also reported. This exchangeability of both the heme fragment and the apofragment of the complex between these two species indicates close similarity of the folding mechanism. Nonetheless, the results also serve to highlight the subtle effects exerted on the folding process by a relatively small number of changes (17%) in the amino acid sequence.

Amino Acid Sequence↗
PubMed1982

Conformational dynamics of a biologically active three-fragment complex of horse cytochrome c.

The conformational dynamics of a biologically active noncovalent complex containing three fragments, ferroheme fragment (1-25)H and apofragments (28-38) and [3H](56-104) [or [3H](39-104)], of horse cytochrome c has been studied with respect to kinetics and thermodynamics of dissociation. The rate of unfolding of the two-fragment complex ferro(1-25)H . (56-104) was also estimated. The results indicate that the ferrous three-fragment complex exhibits a higher frequency of dissociation-association with fragment (28-38) and a lower frequency of overall unfolding-folding at pH 7.0. In the presence of an excess of free (28-38) and below 30 degrees C, unfolding of the ferrous three-fragment complex appears to occur by activation to the transitional state without a large change in conformation, followed by virtually simultaneous dissociation of all three of the fragments [without going through the complex (1-25)H . (56-104), which is a major intermediate for folding]. Above 30 degrees C unfolding via the complex (1-25)H . (56-104) becomes detectable because the equilibrium between the two- and the three-fragment complex is highly temperature dependent. Thus, the relative probabilities of these two different ways of transition for unfolding are modulated by temperature. The observations suggest that the mode of activation of protein and hence the pathway for unfolding may vary depending on temperature. It is also suggested that the interatomic interactions binding the three fragments together in the ordered complex are linked to strengthen each other in the ground state.

Animals↗
PubMed1981

Formation of a cytochrome c-like species from horse apoprotein and hemin catalyzed by yeast mitochondrial cytochrome c synthetase.

Cytochrome c synthetase in yeast mitochondria catalyzes the formation of a yeast cytochrome c-like species from the apoprotein and hemin (Basile, G., DiBello, C., and Taniuchi, H. (1980) J. Biol. Chem. 255, 7181-7191). To test the specificity of this enzyme, 125I-labeled horse apocytochrome c was incubated with the yeast mitochondrial fraction in the presence of hemin, NADPH, and an ethanol extract of the postmitochondrial fraction. A radioactive 125I-labeled cytochrome c-like species was formed in yields of up to 26%. This 125I-labeled species is indistinguishable from horse cytochrome c by ion exchange chromatography (under the conditions which allow separation of horse and yeast cytochrome c), resistance in its reduced form to digestion by trypsin, resistance against autoxidation, reduction by cytochrome b2, and generation of the apoprotein after treatment with silver sulfate and dithiothreitol. With unlabeled horse apoprotein and [59Fe]hemin, the yield of a [59Fe-labeled horse cytochrome c-like species was up to 7% with respect to the apoprotein incubated. The yield of the 59Fe-labeled species was not altered by the addition of unlabeled FeCl3. Conversely, synthesis of the 59Fe-labeled species was not detectable after incubation of yeast mitochondria with unlabeled horse apoprotein, unlabeled hemin, and 59FeCl3. The formation of both 125I- and 59Fe-labeled cytochrome c-like species was sensitive to heat. Thus, we conclude that cytochrome c synthetase catalyzes direct bonding of heme (or hemin) to the apoprotein. Since the amino acid sequences of horse and yeast cytochromes c differ considerably, cytochrome c synthetase may recognize only a limited region(s) of the apoprotein.

Animals↗
PubMed1981

Further study of the conformation of nuclease-(1-126) in relation to intrinsic enzymatic activity.

Nuclease-(1-126), although containing 89% of the amino acid sequence which folds to the ordered structure of nuclease A, is disordered and highly flexible (Taniuchi, H., and Anfinsen, C. B. (1969) J. Biol. Chem. 243, 4778-4786). On the other hand, Sachs et al. (Sachs, D. H., Schechter, A. N., Eastlake, A., and Anfinsen, C. B. (1974) Nature 251, 242-244) have demonstrated intrinsic enzymatic activity for nuclease-(1-126). To attempt to learn whether or not the active population of nuclease-(1-126) has the native conformation, we have examined nuclease-(1-126) with respect to enzymatic kinetics with and without the competitive inhibitor deoxythymidine 3',5'-diphosphate (pdTp), effect of temperature on enzymatic activity, binding of pdTp in the presence of Ca2+ and intrinsic viscosity, Stokes radius, CD, and response to trypsin action in the presence and absence of pdTp and Ca2+. The results indicate that the conformation of nuclease-(1-126) bound with pdTp in the presence of Ca2+ is partially constrained but still highly flexible below 30 degrees C, outside the range of thermal transition exhibited by the ordered elements of nuclease-(1-126). Thus, formation or stabilization of active site of nuclease-(1-126) by binding with ligands is not associated with cooperative folding of the entire polypeptide chain. Considering that nuclease-(1-126) does not bind to nuclease-(127-149) but does to nuclease-(111-149), the results are consistent with the idea that the specific cooperative interactions, providing extra stabilizing energy required for maintaining the polypeptide chain in the ordered state of nuclease A, may be disrupted for nuclease-(1-126) primarily due to cleavage of the peptide bond between residues 126 and 127. Then, it may be thought that binding with ligands does not compensate for this disruption.

Circular Dichroism↗
PubMed1981

Ordered complexes of cytochrome c fragments. Kinetics of formation of the reduced (ferrous) forms.

The kinetics of formation of noncovalently bound ferrous complexes derived from fragments of horse heart cytochrome c have been investigated. When the reactions are initiated by combining ferrous heme fragments with an appropriate apofragment, in the presence of 50 mM imidazole, second order rate processes are observed with rate constants essentially the same as those reported with ferric heme fragments (Parr, G. R., and Taniuchi, H. (1979) J. Biol. Chem. 254, 4836-4842). An additional, probably consecutive, kinetic process is also demonstrated. If imidazole is not present in the reaction buffer, the kinetic profiles are dramatically altered. While this is partially due to aggregation (dimerization) of the ferrous heme fragments, it can nevertheless be demonstrated that the complementation reactions with apofragments are much faster than those observed with the corresponding ferric heme fragments (in the absence of imidazole). These results reflect the effect of the oxidation state of the heme iron on the folding mechanism and, thus, the manifold nature of protein folding pathways. The rate of reduction of productive ferric complexes by sodium ascorbate was investigated and biphasic reactions were found in all cases. The data indicate an equilibrium between two forms of the ferric complexes. The results of an experiment in which the complementation of ferric (1-25)H and (23-104) was carried out in the presence of sodium ascorbate indicate that the intermediate complex (Parr, G. R., and Taniuchi, H. (1980) J. Biol. Chem. 255, 8914-8918) is not reducible by ascorbate. Thus, the increase in oxidation-reduction potential occurring on formation of the productive complex from the unbound heme fragment occurs at a late stage of the overall reaction, possibly coinciding with ligation of methionine 80 to the heme iron.

Animals↗
PubMed1980

Kinetic intermediates in the formation of ordered complexes from cytochrome c fragments. Evidence that methionine ligation is a late event in the folding process.

The reactions of ferric heme-containing fragments with apofragments to form ordered complexes resembling native horse heart cytochrome c have been studied under conditions which resolve the overall process into consecutive second order and first order kinetic steps. In the initial, second order step the two fragments combine to form an intermediate complex which exhibits tryptophan 59 fluorescence quenching similar to native cytochrome c, but which has not yet achieved the native ligation state of the heme iron. The existence of first order processes following the second order step is demonstrated by absorbance changes in the Soret region. the entire absorbance change at 695 nm, relating to ligation of the sulfur atom of methionine 80 to the heme iron, is also associated with these first order processes. Thus, ligation of methionine is a late event in this self ordering of the polypeptide chains. Since the conformational energy is assumed to distinctly decrease in this late process of folding (Parr, G.R., and Taniuchi, H. (1980) J. Biol. Chem. 255, 2616-2623), it would follow that small spatial rearrangements of the polypeptide chains in the late stage of folding (as manifested by the ligation of methionine) are associated with a specific decrease in energy.

Animals↗
PubMed1980

Formation of an iso-1-cytochrome c-like species containing a covalently bonded heme group from the apoprotein by a yeast cell-free system in the presence of hemin.

Incubation of the 125I-labeled apoprotein, prepared from 125I-labeled iso-1-cytochrome c, with a yeast mitochondrial fraction in the presence of hemin, NADPH, and an extract of the postmitochondrial fraction at 32 +/- 1 degree C for 30 min has resulted in formation of cytochrome c-like species in yields of up to 35%. This radioactive synthesized species contains a functional group which responds to reduction with ascorbate and oxidation with K3Fe(CN)6 in that it is resistant in the reduced form and susceptible in the oxidized form to trypsin action in a manner characteristic of native cytochrome c. The functional group cannot be removed from the protein by cold HCl-acetone or 8 M urea treatment. The reduced form of the synthesized species exhibits resistance against autoxidation and the oxidized form can be reduced also by cytochrome b2. The synthesized species exhibits the same compact hydrodynamic volume of native cytochrome c. Treatment with silver sulfate followed by incubation with dithiothreitol converts the synthesized species to the original apoprotein as judged by an increase in the hydrodynamic volume. Thus, the synthesized species is indistinguishable from the original labeled iso-1-cytochrome c by these measurements; i.e. the synthesized species consists of the apoprotein to which heme is covalently attached through the thioether bond(s). The active factor of the mitochondrial fraction is heat-labile. The synthetic activity is strongly dependent on pH with a maximum approximately at pH 7.0. Hemin (or heme) appears to be required for this synthesis. The postmitochondrial fraction is inactive by itself. However, its addition markedly increases the synthetic activity. This factor is heat-stable, soluble in 80% methanol (or 75% ethanol), and insoluble in ethyl ether or ethyl acetate. Addition of NADP(H) (or NAD(H)) also increases the synthetic activity, the reduced form being more effective than the oxidized form. The postmitochondrial factor and the pyridine nucleotides appear to enhance the effect of each other. Thus, it seems that cytochrome c or a cytochrome c-like species is formed from the apoprotein and heme (or hemin) by an enzyme, cytochrome c synthetase, present in mitochondria.

Apoproteins↗