Yeast Cytochrome c Peroxidase Expression in Escherichia coli and Rapid Isolation of Various Highly Pure Holoenzymes

Teske, Jennifer G.; Savenkova, Marina I.; Mauro, J. Matthew; Erman, James E.; Satterlee, James D.

doi:10.1006/prep.2000.1220

Cited by 29 publications

(70 citation statements)

References 30 publications

(35 reference statements)

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“…Heme biosynthesis was stimulated by adding δ-aminolevulinic acid and FeSO 4 to the fermentation mixture (57,60,61). Expression was again induced with IPTG, as above, at a cell density OD 600 ) 0.75.…”

Section: Methodsmentioning

confidence: 99%

“…One-dimensional 1 H NMR spectra (data not shown) reveal essentially identical hyperfine shift patterns for the resting states of all three recombinant enzymes used in this study. In turn, spectra of resting-state recNATCcP and resting-state natWTCcP have been shown to have identical hyperfine shift patterns (57). The similarity in these spectra emphasizes the essentially identical nature of the heme active site in the resting state of all of these enzymes, and reinforces the concept of structurally and functionally intact active sites for both of the perdeuterated enzymes.…”

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confidence: 90%

“…The hyperfine-shifted proton resonances, some of which were previously assigned, are designated by numbers (see Table 3) in Figure 2. Although not shown here, extensive work has revealed that proton NMR spectra (1D and 2D) of recNATCcPCN and natWTCcPCN (40)(41)(42)(43)57) are essentially identical. Figure 2C is a spectrum of recDCcPCN.…”

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confidence: 92%

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Expression, Purification, Characterization, and NMR Studies of Highly Deuterated Recombinant CytochromecPeroxidase

et al. 2001

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Two forms of extensively deuterated S. cerevisiae cytochrome c peroxidase (CcP; EC 1.11.1.5) have been overexpressed in E. coli by growth in highly deuterated medium. One of these ferriheme enzyme forms (recDCcP) was produced using >97% deuterated growth medium and was determined to be approximately 84% deuterated. The second form [recD(His)CcP] was grown in the same highly deuterated medium that had been supplemented with excess histidine (at natural hydrogen isotope abundance) and was also approximately 84% deuterated. This resulted in direct histidine incorporation without isotope scrambling. Both of these enzymes along with the corresponding recombinant native CcP (recNATCcP), which was expressed in a standard medium with normal hydrogen isotope abundance, consisted of 294 amino acid polypeptide chains having the identical sequence to the yeast-isolated enzyme, without any N-terminal modifications. Comparative characterizations of all three enzymes have been carried out for the resting-state, high-spin forms and in the cyanide-ligated, low-spin forms. The primary physical methods employed were electrophoresis, UV-visible spectroscopy, hydrogen peroxide reaction kinetics, mass spectrometry, and (1)H NMR spectroscopy. The results indicate that high-level deuteration does not significantly alter CcP's reactivity or spectroscopy. As an example of potential NMR uses, recDCcPCN and recD(His)CcPCN have been used to achieve complete, unambiguous, stereospecific (1)H resonance assignments for the heme hyperfine-shifted protons, which also allows the heme side chain conformations to be assessed. Assigning these important active-site protons has been an elusive goal since the first NMR spectra on this enzyme were reported 18 years ago, due to a combination of the enzyme's comparatively large size, paramagnetism, and limited thermal stability.

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Section: Methodsmentioning

confidence: 99%

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Expression, Purification, Characterization, and NMR Studies of Highly Deuterated Recombinant CytochromecPeroxidase

et al. 2001

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“…The cloned gene has an identical sequence to that of mature baker's yeast CcP with the exception of the methionine codon required for bacterial expression (34). The N-terminal methionine is removed from the recombinant CcP in this expression system so that there are no N-terminal modifications in the recombinant CcP compared to baker's yeast CcP (35).…”

Section: Starting Clones and Mutagenesismentioning

confidence: 99%

Effect of Single-Site Charge-Reversal Mutations on the Catalytic Properties of Yeast Cytochrome c Peroxidase: Mutations near the High-Affinity Cytochrome c Binding Site

et al. 2007

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Fifteen single-site charge-reversal mutations of yeast cytochrome c peroxidase (CcP) have been constructed in order to determine the effect of localized charge on the catalytic properties of the enzyme. The mutations are located on the front face of CcP, near the cytochrome c binding site identified in the crystallographic structure of the yeast cytochrome c/CcP complex (Pelletier and Kraut (1992) Science 258, 1748-1755. The mutants are characterized by absorption spectroscopy and hydrogen peroxide reactivity at both pH 6.0 and 7.5 and by steady-state kinetic studies using recombinant yeast iso-1 ferrocytochrome c(C102T) as substrate at pH 7.5. Some of the chargereversal mutations cause detectable changes in the absorption spectrum, especially at pH 7.5, reflecting changes in the equilibrium between penta-and hexa-coordinate heme species in the enzyme. An increase in the amount of hexa-coordinate heme in the mutant enzymes correlates with an increase in the fraction of enzyme that does not react with hydrogen peroxide. Steady-state velocity measurements indicate that five of the fifteen mutations cause large increases in the Michaelis constant (R31E, D34K, D37K, E118K, and E290K). These data support the hypothesis that the cytochrome c/CcP complex observed in the crystal is the dominant catalytically active complex in solution.Cytochrome c peroxidase, CcP 1 , is a detoxification enzyme localized between the inner and outer membranes of yeast mitochondria (1). CcP decreases toxic levels of hydrogen peroxide by catalyzing its reduction to water using ferrocytochrome c (2). The catalytic mechanism involves oxidation of the native enzyme by hydrogen peroxide to an enzyme intermediate called CcP Compound I, CcP-I. CcP-I contains two oxidized sites, an oxyferryl Fe(IV) heme group and a tryptophan π-cation radical located within van der Waals distance of the heme. Interaction with, and electron transfer from, ferrocytochrome c reduces CcP-I back to the native state via a second enzyme intermediate, CcP Compound II, CcP-II, completing the catalytic cycle. Since 1980, when the three-dimensional structure of CcP was first reported (3,4), CcP has played an important role in elucidating the structural basis for heme protein reactivity, † This work was supported in part by a grant from the National Institutes of Health (R15 GM59740). * Corresponding author. Phone: (815) . E-mail: jerman@niu.edu. 1 Abbreviations: Mutations in the amino acid sequences of either CcP or cytochrome c are indicated by using the one letter code for the amino acid residue in the wild-type protein, followed by the residue number and the one letter code for the amino acid residue in the mutant protein, i.e., C102T represents a mutant in which a threonine residue replaces the cysteine residue at position 102 of the wildtype protein; CcP, generic abbreviation for cytochrome c peroxidase whatever the source; yCcP, authentic yeast cytochrome c peroxidase isolated from bakers' yeast, Saccharomyces cervisiae; rCcP, recombinant CcP expressed in E. ...

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“…Mutant CcP was obtained from two different recombinant protein systems: CcP(MI) [20] and CcP(eco) [21,22]. CcP(MI) includes two additional amino acids at the N-terminus of the mature protein.…”

Section: Methodsmentioning

confidence: 99%

Effect of active site and surface mutations on the reduction potential of yeast cytochrome c peroxidase and spectroscopic properties of the oxidized and reduced enzyme

DiCarlo

Vitello

Erman

2007

Journal of Inorganic Biochemistry

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The reduction potentials of 22 yeast cytochrome c peroxidase (CcP) mutants were determined at pH 7.0 in order to determine the effect of both heme pocket and surface mutations on the Fe(III)/Fe(II) redox couple of CcP, as well as to determine the range in redox potentials that could be obtained through point mutations in the enzyme. Spectroscopic properties of the Fe(III) and Fe(II) forms of the mutant enzymes are also reported. The mutations include variants in the distal and proximal heme pockets as well as on the enzyme surface and involve single, double, and triple point mutations. A spectrochemical redox titration technique used in this study gave an E o ′ value of −189 mV for yeast CcP compared to a previously reported value of −194 mV determined by potentiometry [C.W. Conroy, P. Tyma, P.H. Daum, J.E. Erman, Biochim. Biophys. Acta 537 (1978) 62-69.] Both positive and negative shifts in the reduction potential from that of the wild-type enzyme were observed, spanning a range of 113 mV. The His-52 → Asn mutation gave the most negative potential, −259 mV, while a triple mutant in which the three distal pocket residues, Arg-48, Trp-51, and His-52, were all converted to leucine residues gave the most positive potential, −146 mV.

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Yeast Cytochrome c Peroxidase Expression in Escherichia coli and Rapid Isolation of Various Highly Pure Holoenzymes

Cited by 29 publications

References 30 publications

Expression, Purification, Characterization, and NMR Studies of Highly Deuterated Recombinant CytochromecPeroxidase

Expression, Purification, Characterization, and NMR Studies of Highly Deuterated Recombinant CytochromecPeroxidase

Effect of Single-Site Charge-Reversal Mutations on the Catalytic Properties of Yeast Cytochrome c Peroxidase: Mutations near the High-Affinity Cytochrome c Binding Site

Effect of active site and surface mutations on the reduction potential of yeast cytochrome c peroxidase and spectroscopic properties of the oxidized and reduced enzyme

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