Search

A system for the construction of polymeric peptides in Escherichia coli was utilized to prepare a library of plasmids coding for tandem repeats of the Neurospora crassa metallothionein gene. Selected oligomeric metallothionein clones were expressed and targeted to the periplasm as a fusion with the maltose-binding protein. Bacterial cells harbouring the expressed oligopeptides were characterized for their ability to bind 109Cd2+. The metal-binding ability was enhanced for all the oligomeric constructs tested and, in the best case, a 6.5-fold increased capacity for metal uptake was achieved with cells expressing a tandem 9-mer in comparison with cells expressing a monomer. Plateauing of the metal uptake ability occurred at between six and nine tandem repeats, possibly due to a combination of lowered translation levels, inefficient export and prematurely terminated translation products. The overall enhancement of the heavy metal removal capacity was approximately 65-fold relative to non-recombinant cells. The use of this strategy for the design and expression of de novo polypeptides containing multiple functional domains for use in bioremediation is discussed.

The gene coding for the Neurospora crassa metallothionein protein was chemically synthesized and cloned into the fusion expression vectors pMal-c and pMal-p. Cell-fractionation experiments demonstrated the proper localization of the pMal-c and pMal-p- expressed proteins to the cytosol and periplasm of the bacteria respectively. Control bacteria as well as the recombinant bacteria producing the metallothionein protein were incubated with solutions of 109Cd at concentrations of 0.2 microM, 1 microM, and 10 microM. The recombinant bacteria were able to accumulate significantly more 109Cd than control bacteria at all concentrations tested. Cadmium accumulation was rapid and highly selective. Maximum uptake was achieved at a pH of 7.0, with lower accumulation at lower or higher pH values. The pH-dependent uptake of cadmium by the recombinant bacteria was exploited to strip off the bound cadmium from the recombinant bacteria and to regenerate most of the cadmium-binding sites. These observations suggest the potential for using a metallothionein-based biosorbent for certain heavy-metal removal applications.

The thermodynamics of binding of fluoride and cyanide to cytochrome c peroxidase (CCP) and its Asn-82-->Asp mutant (D82CCP) in phosphate and acetate buffer at an ionic strength of 0.15 mol.kg-1 from pH 5.0 to 7.1 were investigated by titration calorimetry at 289 and 297 K. The binding reactions are enthalpically driven. The fluoride-binding constants determined from the titration calorimetry results were in agreement with those determined from difference-spectroscopy measurements. For cyanide binding to CCP at 297.9 K, the binding constant decreased from 8.95 (+/- 0.83) x 10(5) M-1 at pH 7.0 to 4.04(+/- 0.23) x 10(5) M-1 at pH 5.0, and the binding enthalpy increased from -57.2 +/- 1.4 kJ.mol-1 at pH 7.0 to -48.6 +/- 1.8 kJ.mol-1 at pH 5.0. For fluoride binding to CCP, the binding constant increased from 8.41(+/- 0.54) x 10(3) M-1 at pH 7.0 to 3.11(+/- 0.09) x 10(5) M-1 at pH 5.0 and the binding enthalpy increased from -71.9 +/- 1.1 kJ.mol-1 at pH 7.0 to -67.0 +/- 1.9 kJ.mol-1 at pH 5.0. The binding enthalpies for D82CCP were about the same as those for CCP. However, the binding constants for cyanide and fluoride to D82CCP were respectively a factor of two less and at least an order of magnitude less than the corresponding binding constants of CCP. Decreased ligand-binding strength in the D82CCP mutant is thus entirely due to entropic effects.

The bimolecular reaction between Escherichia coli-produced cytochrome-c peroxidase (CcP(MI)) and hydrogen peroxide is identical to that of native yeast cytochrome-c peroxidase (CcP) and hydrogen peroxide in the neutral pH region. Both enzymes have pH-independent bimolecular rate constants of 46 microM-1.s-1 for the reaction with hydrogen peroxide. A second mutant enzyme, E. coli-produced cytochrome-c peroxidase mutant with phenylalanine at position 191 (CcP(MI, F191)), has a pH-independent bimolecular rate constant for the hydrogen peroxide reaction of 65 microM-1.s-1, 40% larger than for CcP or CcP(MI). The initial peroxide-oxidation product of CcP(MI, F191) is an oxyferryl porphyrin pi-cation radical intermediate in contrast to the oxyferryl amino-acid radical intermediate formed upon oxidation of CcP or CcP(MI) with hydrogen peroxide. The reactions of all three enzymes with hydrogen peroxide are pH-dependent in KNO3-containing buffers. The reactions are influenced by an ionizable group, which has an apparent pKa of 5.4 in all three enzymes. The enzymes react with hydrogen peroxide when the ionizable group is unprotonated. Both CcP(MI) and CcP(MI, F191) have slightly smaller pH stability regions compared to CcP as assessed by the hydrogen peroxide titer and spectral analysis. The alteration in structural stability must be attributed to differences in the primary sequence between CcP and CcP(MI) which occur at positions -2, -1, 53 and 152.

Using site-directed mutagenesis, a double mutant in yeast cytochrome c peroxidase (CCP) has been constructed where the proximal ligand, His175, has been converted to glutamine and the neighboring Trp191 has been converted to phenylalanine. The refined 2.4-A crystal structure of the double mutant shows that the Gln175 side chain is within coordination distance of the heme iron atom and that Phe191 occupies the same position as Trp191 in the native enzyme with very little rearrangement outside the immediate vicinity of the mutations. Consistent with earlier work, we find that the single mutant, His175--Gln, is fully active under steady state assay conditions and that as reported earlier (Mauro et al., 1988), the Trp191--Phe mutant exhibits only0.05% activity. However, the double mutant, His175--Gln/Phe191--Phe, exhibits 20% wild type activity. Since it is known that the Trp191--Phe mutant is inactive because it can no longer transfer electrons from ferrocytochrome c, changing the nature of the proximal ligand is able to restore this activity. These results raise interesting questions regarding the mechanism of interprotein electron transfer reactions.

To investigate the molecular basis for the 100-fold slower rate of CO dissociation in ferrous peroxidases relative to myoglobin, CO dissociation rates were measured as a function of pH in the cloned cytochrome c peroxidase from yeast [CCP(MI)] and in several mutants in the heme binding pocket prepared by site-directed mutagenesis. The mutants included Asp 235----Asn; Arg 48----Lys, Leu; and His 181----Gly. Changes in the absorption spectrum with pH are consistent with conversion of the CO-ferrous CCP(MI) complex from acidic to alkaline forms by a two-proton cooperative ionization, with an apparent pKa = 7.6, analogous to that described for CCP from bakers' yeast [Iizuka, T., Makino, R., Ishimura, Y.,Yonetani, T. (1985) J. Biol. Chem. 260, 1407-1412]. The rate of CO dissociation (koff) was increased 11-fold (from 0.7 x 10(-4) to 8.0 x 10(-4) s-1) by conversion of the acidic to the alkaline form. Analogous acidic and alkaline forms of the CO complex were also observed in the mutants of CCP(MI) examined here. In the acidic form, koff was increased 5- and 20-fold when Arg 48 was replaced with Lys and Leu, respectively, while in the acidic form of mutants that possess Arg 48, koff was similar to that observed in CCP(MI). Conversion of the CO complex from the acidic to alkaline form increased koff in all the mutants, and the pH-dependent increase in koff correlated with a two-proton cooperative ionization, except in the case of His 181----Gly. In this mutant, pH-dependent increase in koff correlated with a single-proton ionization, implicating His 181 as one of the two residues that is deprotonated in the conversion of CO-ferrous CCP(MI) from acidic to alkaline forms. Only a 2.5-fold variation was observed for koff between the alkaline form of CCP(MI) and the Arg 48----Leu mutant, suggesting that the influence of Arg 48 on the rate of CO dissociation is decreased in the alkaline form by a conformational change.(ABSTRACT TRUNCATED AT 400 WORDS)

CO recombination to the cloned cytochrome c peroxidase [CCP(MI)] and mutants of CCP(MI) prepared by site-directed mutagenesis was examined as a function of pH by flash photolysis. The mutants examined included distal Arg 48----Leu, Lys; proximal Asp 235----Asn; and His 181----Gly. At alkaline pH, ferrous CCP(MI) was converted to a hexacoordinate form by a cooperative two-proton ionization, apparent pK(a) = 8.0. This change was observed in all of the mutants, although in the His 181----Gly mutant, the conversion to the hexacoordinate form was the result of a single-proton ionization, implicating His 181 as one of the two residues deprotonated in this isomerization. The pH-dependent conversion of CO ferrous CCP(MI) from acidic to alkaline forms was also observed and was similar to that reported for cytochrome c peroxidase from bakers' yeast [Iizuka, T., Makino, R., Ishimura, Y.,Yonetani, T. (1985) J. Biol. Chem. 260, 1407-1412]. Photolysis of the acidic form of the CO complex of CCP(MI) produces a kinetic form of the ferrous enzyme (form A) which exhibits the slow rate of CO recombination (l1' approximately 10(3) M-1 s-1) characteristic of peroxidases, while photolysis of the alkaline form of the CO complex produces a second kinetic form (form B), which exhibits a much faster rate of recombination (l2' approximately 10(5) M-1 s-1). Kinetic forms analogous to forms A and B were observed in all of the mutants examined. A third kinetic form (form B*) with a bimolecular rate constant l3' approximately 10(6) M-1 s-1 was also observed in the mutants at alkaline pH. Although the pH dependence for the conversion of form A to form B with increasing pH was altered by changes in the local heme environment, the rate of CO recombination by the respective forms was not dramatically altered in the mutants. Transient spectra of the reaction of CO with ferrous CCP(MI) after photolysis show that equilibrium between penta- and hexacoordinate ferrous enzyme is rapid relative to CO recombination. The presence of the internal sixth ligand has no discernible effect on the observed rate of recombination, however. The results presented indicate that in CCP(MI) the rate of ligand binding is determined primarily by isomerization of the protein from a closed conformation at acidic pH to an open conformation at alkaline pH and that polar effects of proximal Asp 235 and distal Arg 48 are of minor significance in the rate of CO recombination in both conformations.

A photolabile reagent, p-azidophenacyl-DL-thiocarnitine, was synthesized and tested as a photoaffinity label for carnitine acetyltransferase (EC 2.3.1.7) from pigeon breast. p-Azidophenacyl-DL-thiocarnitine is an active-site-directed reagent for this acetyltransferase, since it is a competitive inhibitor (Ki 10 microM) versus carnitine. U.v. irradiation of a mixture of p-azidophenacyl-DL-thiocarnitine and enzyme produces irreversible inhibition. Acetyl-DL-carnitine protects the enzyme from inhibition by photoactivated p-azidophenacyl-DL-thiocarnitine. In the presence of 30 mM-2-mercaptoethanol as a scavenger, the relationship between loss of activity and photoincorporation of reagent suggests that one molecule of reagent is incorporated per molecule of inhibited enzyme. However, peptide maps of enzyme labelled with p-azidophenacyl[14C]thiocarnitine indicate that several (about six) tryptic peptides (of a possible 60-65) are modified. The presence of 5 mM-acetyl-DL-carnitine significantly decreases the incorporation of reagent in each labelled tryptic peptide.

A difference Fourier map shows small structural perturbations on oxidation of cytochrome c peroxidase (CCP) to its semi-stable intermediate, compound I. Least-squares refinement of both CCP and compound I quantifies these perturbations and suggests that the radical site may be on the distal side of the heme, since that is where most of the small movements occur. Several engineered mutants of CCP were created in an attempt to assess the function of various side chains, among them Trp-51----Phe, Trp-191----Phe and His-181----Gly. X-ray structures of the mutant CCP's confirm that only minimal changes are caused by these substitutions. Preliminary examination of the mutants' kinetic properties show that Trp-51 is not the radical site; that Trp-191 has an important enzymic function; and that His-181 is not essential for electron transfer, but probably has some more indirect role. The locus of the radical in compound I, however, remains to be established.

Peroxide oxidation of a mutant cytochrome c peroxidase, in which Trp-191 has been replaced by Phe through site-directed mutagenesis, produces an oxidized intermediate whose stable UV/visible absorption spectrum is very similar to that of compound I of the native yeast enzyme. This spectrum is characteristic of an oxyferryl, Fe(IV), heme. Stopped-flow studies reveal that the reaction between the mutant enzyme and hydrogen peroxide is biphasic with the transient formation of an intermediate whose absorption spectrum is quite distinct from that of either the native ferric enzyme or the final product. Rapid spectral scanning of the intermediate provides a spectrum characteristic of an oxyferryl porphyrin pi-cation-radical species. At pH 6, 100 mM ionic strength, and 25 degrees C, the rate constant for formation of the oxyferryl pi-cation radical has a lower limit of 6 X 10(7) M-1 s-1 and the rate of conversion of the transient intermediate to the final oxidized product is 51 +/- 4 s-1. Evidence is presented indicating that Trp-191 either is the site of the radical in CcP compound I or is intimately involved in formation of the radical.