Studies on NADPH-cytochrome c reductase. II. Steady-state kinetic properties of the crystalline enzyme from ale yeast

From a study of the steady-state kinetics (at pH 7.6, 30 degrees C) of the reduction of cytochrome c, a 'ping-pong' mechanism may be postulated for the crystalline NADPH-cytochrome c reductase from ale yeast, Saccharomyces cerevisiae [1], a result derivable from a three-substrate ordered system with a rapid equilibrium random sequence in substrates, NADPH and FAD, followed by reactions of the third substrate, Cyt C3+. On this basis, estimates for the kinetic parameters were made together with the inhibitor dissociation constants for NADP+ (competitive with respect to NADPH as variable substrate, but noncompetitive with respect to cytochrome c3+ as the variable substrate). A noncompetitive type of inhibition was also found for cytochrome c2+ with NADPH as variable substrate, in confirmation of the proposed mechanism. With 2,6-dichloroindophenol as the acceptor, in place of cytochrome c3+, a value for KNADPH could be estimated which agreed with that estimated above, with cytochrome c3+ as the acceptor, again, in confirmation of the postulated mechanism. The reactions with molecular O2 catalyzed by the enzyme with NADPH as the reductant have been studied polarographically, and its Km for O2 estimated to be about 0.15 mmol/l at pH 7.6, 25 degrees C. The product of the reaction appears to be H2O2, which acts as a noncompetitive inhibitor for NADPH (Ki = 0.5 mmol/l), and tentatively an enzyme ternary complex containing oxygen and FADoh (semiquinone of FAD) may be assumed to be the kinetically important intermediate, which may be postulated to be in quasi-equilibrium with an enzyme ternary complex containing Oo2 (superoxide) and FAD.     

Studies on NADPH-cytochrome c reductase I: Isolation and several properties of the crystalline enzyme from ale yeast.

NADPH-cytochrome c reductase has been isolated from a top-fermenting ale yeast, Saccharomyces cerevisiae (Narragansett strain), after ca. a 240-fold purification over the initial extract of an acetone powder, with a final specific activity (at pH 7.6, 30 °C) of ca. 150 μmol cytochrome c reduced min^?1mg^?1 protein. The preparation appears to be homogeneous by the criteria of: sedimentation velocity; electrophoresis on cellulose acetate in buffers above neutrality; and by polyacrylamide gel electrophoresis. Although the reductase appeared to partially separate into species “A” and “B” on DEAE-cellulose at pH 8.8, the two species have proven to be indistinguishable electrophoretically (above pH 8) and by sedimentation. By sedimentation equilibrium at 20 °C, a molecular weight of ca. 6.8 (± 0.4) × 10⁴ was obtained with use of a $\text{V}\text{?}{}_{\mathrm{20\; °}}\text{= 0.741}$ calculated from its amino acid composition. After disruption in 4 m guanidinium chloride- 10 mm dithioerythritol- 1 mm EDTA, pH 6.4 at 20 °C, an $\text{M}\text{?}{}_{\mathrm{<mtext>r</mtext>}}$ of 3.4 (± 0.1) × 10⁴ resulted, which points to a subunit structure of two polypeptide chains per mole. Confirmatory evidence of the two-subunit structure with similar, if not identical, polypeptide chains was obtained by polyacrylamide gel electrophoresis in dodecyl-sulfate, after disruption in 4 m urea and 2% sodium dodecyl sulfate, and yielded a subunit molecular weight of ca. 4 × 10⁴. Sulfhydryl group titration with 4,4′-dithiodipyridine under acidic conditions revealed one sulfhydryl group per monomer, which apparently is necessary for the catalytic reduction of cytochrome c. NADPH, as well as FAD, protects this-SH group from reaction with 5,5′-dithiobis (2-nitrobenzoate). The visible absorption spectrum of the oxidized enzyme (as prepared) has absorption maxima at 383 and 455 nm, typical of a flavoprotein. Flavin analysis (after dissociation by thermal denaturation of the “A” protein) conducted fluorometrically, revealed the presence of 2.0 mol of FAD per 70,000 g, in confirmation of the deduced subunit structure. The identity of the FAD dissociated from either “A” or “B” protein was confirmed by recombination with apo-d-amino acid oxidase and by thin-layer chromatography. A kinetic approach was used to estimate the dissociation constant for either FAD or FMN (which also yields a catalytically active enzyme) to the apoprotein reductase at 30 °C and pH 7.6 (0.05 m phosphate) and yielded values of 4.7 × 10^?8m for FAD and 4.4 × 10^?8m for FMN.     

Mutational analysis of the yeast coenzyme QH2-cytochrome c reductase complex

The synthesis of cytochrome b in yeast depends on the expression of both mitochondrial and nuclear gene products that act at the level of processing of the pre-mRNA, translation of the mRNA, and maturation of the apoprotein during its assembly with the nuclear-encoded subunits of coenzyme QH2-cytochrome c reductase. Previous studies indicated one of the nuclear genes (CBP2) to code for a protein that is needed for the excision of the terminal intervening sequence from the pre-mRNA. We show here that the intervening sequence can promote its own excision in the presence of high concentrations of magnesium ion (50 mM), but that at physiological concentrations of the divalent cation (5 mM), the splicing reaction requires the presence of the CBP2-encoded product. These results provide strong evidence for a direct participation of the protein in splicing, most likely in stabilizing a splicing competent structure in the RNA. The conversion of apocytochrome b to the functional cytochrome has been examined in mutants lacking one or multiple structural subunits of the coenzyme QH2-cytochrome c reductase complex. Based on the phenotypes of the different mutants studied, the following have been concluded. (i) The assembly of catalytically active enzyme requires the synthesis of all except the 17 kDa subunit. (ii) Membrane insertion of the individual subunits is not contingent on protein-protein interactions. (iii) Assembly of the subunits occurs in the lipid bilayer following their insertion. (iv) The attachment of haem to apocytochrome b is a late event in assembly after an intermediate complex of the structural subunits has been formed. This complex minimally is composed of apocytochrome b, the non haem iron protein and all the non-catalytic subunits except for the 17 kDa core 3 subunit.     

L-Lactate cytochrome c reductase: rapid kinetic studies of electron transfers within the flavocytochrome b2-cytochrome c assembly

This study is part of a series aimed at the characterization of individual steps of electron transfer taking place between prosthetic flavin, heme b2, heme c within active sites and complexes. After rapid mixing of ferricytochrome c with partially reduced flavocytochrome b2, the reaction is followed at the level of two reactants, cytochrome b2 and cytochrome c. In order to define the proper reactivity of flavosemiquinone, conditions under which this form is highly stabilized (presence of pyruvate) have been chosen. With the help of simulations, it has been possible to characterize a rapid step of electron transfer from cytochrome b2 to cytochrome c within a complex (at approx. 70% saturation) and a slow step k = 5 s-1 assigned to cytochrome b2 reduction by flavosemiquinone within the active site of the pyruvate-liganded enzyme.     

Purification and characterization of a novel NADPH(NADH)-dependent glyoxylate reductase from spinach leaves. Comparison of immunological properties of leaf glyoxylate reductase and hydroxypyruvate reductase.

A novel reductase displaying high specificity for glyoxylate and NADPH was purified 3343-fold from spinach leaves. The enzyme was found to be an oligomer of about 125 kDa, composed of four equal subunits of 33 kDa each. A Km for glyoxylate was about 14-fold lower with NADPH than with NADH (0.085 and 1.10 mM respectively), but the maximal activity, 210 mumol/min per mg of protein, was similar with either cofactor. Km values for NADPH and NADH were 3 and 150 microM respectively. Optimal rates with either NADPH or NADH were found in the pH range 6.5-7.4. The enzyme also showed some reactivity towards hydroxypyruvate with rates less than 2% of those observed for glyoxylate. Results of immunological studies, using antibodies prepared against either glyoxylate reductase or spinach peroxisomal hydroxypyruvate reductase, suggested substantial differences in molecular structure of the two proteins. The high rates of NADPH(NADH)-glyoxylate reductase in crude leaf extracts of spinach, wheat and soya bean (30-45 mumol/h per mg of chlorophyll) and its strong affinity for glyoxylate suggest that the enzyme may be an important side component of photorespiration in vivo. In leaves of nitrogen-fixing legumes, this reductase may also be involved in ureide breakdown, utilizing the glyoxylate produced during allantoate metabolism.     

Studies on cytochrome c. VI. Synthesis of the protected pentadecapeptide (sequence 64-81) of Baker's yeast iso-1-cytochrome c

The syntheses are reported of two N-benzloxycarbonylpeptide tert-butoxycarbonylhydrazides corresponding to the amino acid sequences 67–74 and 75–81 of the baker's yeast iso-1-cytochrome c and their subsequent assembly to the N-benzyloxycarbonylpentadecapeptide tert-butoxycarbonylhydrazide corresponding to the sequence 67–81 of the protein.     

Studies on cytochrome c. VII. Synthesis of the protected undecapeptide (sequence 82-92) of Baker's yeast iso-1-cytochrome c

The synthesis is described of the N-benzyloxycarbonylundecapeptide tert-butoxycarbonylhydrazide corresponding to the sequence 82–92 of baker's yeast iso-I-cytochrome c.     

Studies on cytochrome c. 8. Synthesis of the protected hexadecapeptide (sequence 93-108) of Baker's yeast iso-1-cytochrome c

The synthesis is described of the protected hexadecapeptide corresponding to the C-terminal sequence 93–108 of baker's yeast iso-I-cytochrome c. The cysteine residue in position 107 of the natural sequence has been substituted by a threonine residue. The rationale of this substitution as well as the synthetic route to the preparation of the hexadecapeptide derivative is discussed.     

Physical and kinetic properties of a pyridoxal reductase purified from bakers' yeast

Pyridoxine dehydrogenase (1.1.1.65) (pyridoxal reductase), purified to homogeneity from baker's yeast, is a monomer of Mr approximately 33,000. It catalyzes the reversible oxidation of pyridoxine by NADP to yield pyridoxal and NADPH; equilibrium lies far in the direction of pyridoxine formation (Keq approximately 1.4 X 10(11) l/mol at 25 degrees C). Reduction of pyridoxal occurs most rapidly at pH 6.0-7.0; oxidation of pyridoxine is optimal at pH 8.6. NAD and NADH do not replace NADP and NADPH as substrates; pyridoxine, pyridoxal and pyridoxal 5'-phosphate are the only naturally occurring cosubstrates found. Several other aromatic aldehydes also are reduced, but substrate specificity and other properties of the enzyme distinguish it clearly from other alcohol dehydrogenases or aldehyde reductases. Between pH 6.3 and 7.1 (the intracellular pH of yeast), V/Km with pyridoxal and NADPH as substrates is greater than 600 times that observed with pyridoxine and NADPH as substrates is greater than 600 times that observed with pyridoxine and NADP as substrates. These and other considerations strongly indicate that the dehydrogenase functions in vivo to reduce pyridoxal to pyridoxine, which is the preferred substrate for pyridoxal (pyridoxine) kinase in yeast.     

Studies on cytochrome c. V. Synthesis of the protected decapeptide (sequence 57-66) of Baker's yeast iso-1-cytochrome c

The synthesis is described of the N-benzyloxycarbonyldecapeptide tert-butoxycarbonylhydrazide, which corresponds to the sequence 57–66 of baker's yeast iso-1-cytochrome c. The peptide derivative was synthesized coupling two smaller subunits via the Rudinger modified azide procedure.     

Studies on the CoQH2-cytochrome c reductase segment of the respiratory chain of yeast mitochondria, using mutants of the cytochrome b split gene

Our work relating to the role of cytochrome b in the CoQH2-cytochrome c reductase segment of the respiratory chain of S. cerevisiae mitochondria is reviewed here and new results are reported. The results concerning the structure-function relationship of cytochrome b in this complex, analyzed within the framework of the eight transmembrane alpha helice cytochrome b folding model, agree with the following features of the proton motive Q cycle (or SQ cycle): i) the antimycin A and myxothiazol binding domains are located on opposite sides of the inner mitochondrial membrane; and ii) the antimycin A binding domain is associated with the b562 domain, the myxothiazol domain with the b565 domain. These results were obtained from structural data derived from amino-acid sequence studies on mit- mutants and from biochemical studies of these mutants. However, functional studies are reported here that are not in agreement with the following features of the above models: i) the serial arrangement of the two hemes of cytochrome b and ii) the isolation of cytochrome b from redox changes with the couple fumarate/succinate in the presence of antimycin A and myxothiazol.     

Reductive inactivation of yeast glutathione reductase by Fe(II) and NADPH

Glutathione reductase (GR) carries out the enzymatic reduction of glutathione disulfide (GSSG) to its reduced form (GSH) at the expense of the reducing power of NADPH. Previous studies have shown that GR from several species is progressively inactivated in the presence of NADPH, but that the mechanism of inactivation (especially in the presence of metals) has not been fully elucidated. We have investigated the involvement of iron ions in the inactivation of yeast (Saccharomyces cerevisiae) GR in the presence of NADPH. Even in the absence of added iron, inactivation of GR was partly blocked by the iron chelators, deferoxamine and ortho-phenanthroline, suggesting the involvement of trace amounts of contaminating iron in the mechanism of inhibition. Exogenously added antioxidants including ethanol, dimethylsulfoxide and 2-deoxyribose did not protect GR against NADPH-induced inactivation, whilst addition of exogenous Fe(II) (but not Fe(III)) potentiated the inactivation. Moreover, removal of oxygen from the medium led to increased inhibition of GR, whereas pre-incubation of the Fe(II)-containing medium for 30 min under normoxic conditions prior to the addition of GR abolished the enzyme inactivation by NADPH. Under these pre-incubation conditions, Fe(II) is fully oxidized to Fe(III) within 1 min. Furthermore, GR that had been previously inactivated in the presence of Fe(II) plus NADPH could be partially reactivated by treatment with ortho-phenanthroline and deferoxamine. In contrast, Fe(III) had no effect on GR reactivation. Together, these results indicate that yeast GR is inactivated by a reductive mechanism mediated by NADPH and Fe(II). According to this mechanism, GR is diverted from its normal redox cycling by the generation of an inactive reduced enzyme form in which both the FAD and thiol groups at the active site are likely in a reduced state.     

The opposite effect of bivalent cations on cytochrome b5 reduction by NADH:cytochrome b5 reductase and NADPH:cytochrome c reductase.

The effects of bivalent cations on cytochrome b5 reduction by NADH:cytochrome b5 reductase and NADPH:cytochrome c reductase were studied with the proteinase-solubilized enzymes. Cytochrome b5 reduction by NADH:cytochrome b5 reductase was strongly inhibited by CaCl2 or MgCl2. When 1.2 microM-cytochrome b5 was used, the concentrations of CaCl2 and MgCl2 required for 50% inhibition (I50) were 8 and 18 mM respectively. The inhibition was competitive with respect to cytochrome b5. The extent of inhibition by CaCl2 or MgCl2 was much higher than that by KCl or other alkali halides. In contrast, cytochrome b5 reduction by NADPH:cytochrome c reductase was extremely activated by CaCl2 or MgCl2. In the presence of 5 mM-CaCl2, the activity was 24-fold higher than control when 4.4 microM-cytochrome b5 was used. The magnitude of activation by CaCl2 was 2-3-fold higher than that by MgCl2. The activation by these salts was much higher than that by KCl, indicating that bivalent cations play an important role in this activation. The mechanisms of inhibition and activation by bivalent cations of cytochrome b5 reduction by these two microsomal reductases are discussed.     

Studies on cytochrome c. Part III. Synthesis of the protected heneicosapeptide (sequence 24–44) of Baker's yeast iso-1-cytochrome c

Syntheses are described for two N-benzyloxycarbonylpeptide tert-butoxycarbonylhydrazides which correspond to positions 24–34 and 35–44, respectively, of the primary structure of baker's yeast iso-1-cytochrome c. The two peptide derivatives were coupled via the azide procedure to form the N-benzyloxycarbonylheneicosapeptide tert-butoxycarbonylhydrazide (sequence 24–44).     

Studies on cytochrome c. Part IV. Synthesis of the protected dodecapeptide (sequence 45–56) of Baker's yeast iso-1-cytochrome c

The synthesis is described for the N-benzyloxycarbonyldodecapeptide tert-butoxycarbonylhydrazide corresponding to the sequence 45–56 of baker's yeast iso-1-cytochrome c by fragment condensation using the Rudinger modified azide procedure for the assembly of the small subunits.     

Studies on cytochrome c. Part II. Synthesis of the protected heptapeptide (sequence 17–23) of Baker's yeast iso-1-cytochrome c

Synthesis is described for the N-o-nitrophenylsulfenylheptapeptide tert-butoxycarbonylhydrazide corresponding to positions 17–23 of the amino acid sequence of baker's yeast iso-1-cytochrome c. Moreover a new method of selective removal of the S-acetamidomethyl group for analytical and preparative purpose is reported.     

Transient-state and steady-state kinetic studies of the mechanism of NADH-dependent aldehyde reduction catalyzed by xylose reductase from the yeast Candida tenuis

Microbial xylose reductase, a representative aldo-keto reductase of primary sugar metabolism, catalyzes the NAD(P)H-dependent reduction of D-xylose with a turnover number approximately 100 times that of human aldose reductase for the same reaction. To determine the mechanistic basis for that physiologically relevant difference and pinpoint features that are unique to the microbial enzyme among other aldo/keto reductases, we carried out stopped-flow studies with wild-type xylose reductase from the yeast Candida tenuis. Analysis of transient kinetic data for binding of NAD(+) and NADH, and reduction of D-xylose and oxidation of xylitol at pH 7.0 and 25 degrees C provided estimates of rate constants for the following mechanism: E + NADH right arrow over left arrow E.NADH right arrow over left arrow E.NADH + D-xylose right arrow over left arrow E.NADH.D-xylose right arrow over left arrow E.NAD(+).xylitol right arrow over left arrow E.NAD(+) right arrow over left arrow E.NAD(+) right arrow over left arrow E + NAD(+). The net rate constant of dissociation of NAD(+) is approximately 90% rate limiting for k(cat) of D-xylose reduction. It is controlled by the conformational change which precedes nucleotide release and whose rate constant of 40 s(-)(1) is 200 times that of completely rate-limiting E.NADP(+) --> E.NADP(+) step in aldehyde reduction catalyzed by human aldose reductase [Grimshaw, C. E., et al. (1995) Biochemistry 34, 14356-14365]. Hydride transfer from NADH occurs with a rate constant of approximately 170 s(-1). In reverse reaction, the E.NADH --> E.NADH step takes place with a rate constant of 15 s(-1), and the rate constant of ternary-complex interconversion (3.8 s(-1)) largely determines xylitol turnover (0.9 s(-1)). The bound-state equilibrium constant for C. tenuis xylose reductase is estimated to be approximately 45 (=170/3.8), thus greatly favoring aldehyde reduction. Formation of productive complexes, E.NAD(+) and E.NADH, leads to a 7- and 9-fold decrease of dissociation constants of initial binary complexes, respectively, demonstrating that 12-fold differential binding of NADH (K(i) = 16 microM) vs NAD(+) (K(i) = 195 microM) chiefly reflects difference in stabilities of E.NADH and E.NAD(+). Primary deuterium isotope effects on k(cat) and k(cat)/K(xylose) were, respectively, 1.55 +/- 0.09 and 2.09 +/- 0.31 in H(2)O, and 1.26 +/- 0.06 and 1.58 +/- 0.17 in D(2)O. No deuterium solvent isotope effect on k(cat)/K(xylose) was observed. When deuteration of coenzyme selectively slowed the hydride transfer step, (D)()2(O)(k(cat)/K(xylose)) was inverse (0.89 +/- 0.14). The isotope effect data suggest a chemical mechanism of carbonyl reduction by xylose reductase in which transfer of hydride ion is a partially rate-limiting step and precedes the proton-transfer step.     

Purification and characterization of a novel NADPH(NADH)-dependent hydroxypyruvate reductase from spinach leaves. Comparison of immunological properties of leaf hydroxypyruvate reductases.

5'-Deoxy-5'-methylthioadenosine, a by-product of polyamine synthesis, can support the growth of Raji cells in a methionine-free medium, but not the growth of CCL39 cells, although these cells are also able to incorporate radiolabelled 5'-deoxy-5'-methylthioadenosine (MeSAdo) into methionine, S-adenosyl-L-methionine (AdoMet) and proteins [Christa, Kersual, Augé & Pérignon (1986) Biochem. Biophys. Res. Commun. 135, 131-138]. We first tested the hypothesis of a toxic effect of MeSAdo in the conditions of growth experiments: we could not demonstrate any toxic effect of MeSAdo on the synthesis of macromolecules, nor any toxicity mediated by polyamines or pyrimidine starvation, and we found that the growth of CCL39 cells was strictly dependent on the supply of exogenous methionine. We then tried to determine whether the ability of CCL39 cells to metabolize MeSAdo to methionine and AdoMet was modulated by the proliferation state of CCL39 cells, which is dependent on the supply of exogenous methionine. Studies of the incorporation of radiolabelled MeSAdo show that: (i) the total synthesis of methionine from MeSAdo is twice as high in subconfluent cells (grown in 100 microM-methionine) as in resting cells (cultured in 0 microM-methionine); (ii) the incorporation into proteins does not parallel the total protein synthesis, and the methionine derived from MeSAdo mostly flows out of the cell; (iii) addition of methionine to resting cells immediately leads to a transient and marked increase in metabolism of MeSAdo to AdoMet, presumably reflecting the rapid replenishment of the AdoMet pool of the cells. Taken together, these results suggest that the methionine derived from MeSAdo is preferentially used to synthesize AdoMet rather than proteins, and that this synthesis of AdoMet depends on the ability of the CCL39 cells to grow, and hence on the supply of exogenous methionine. It is proposed that, in CCL39 cells, the metabolic pathway leading from MeSAdo (a by-product of polyamine synthesis) to methionine and to AdoMet (a precursor of polyamine synthesis) is part of a metabolic cycle the activity of which depends, like polyamine synthesis itself, on cell proliferation.