Examination of the activity of carboxyl-terminal chimeric constructs of human and yeast ferrochelatases

Amy Elizabeth Medlock, Harry A. Dailey

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

Insertion of ferrous iron into protoporphyrin IX is catalyzed by ferrochelatase (EC 4.99.1.1). Human and Schizosaccharomyces pombe forms of ferrochelatase contain a [2Fe-2S] cluster with three of the four coordinating cysteine ligands located within the 30 carboxyl-terminal residues. Saccharomyces cerevisiae ferrochelatase contains no cluster, but has comparable activity. Truncation mutants of S. cerevisiae lacking either the last 37 or 16 amino acids have no enzyme activity. Chimeric mutants of human, S. cerevisiae, and Sc. pombe ferrochelatase have been created by switching the terminal 10% of the carboxy end of the enzyme. Site-directed mutagenesis has been used to introduce the fourth cysteinyl ligand into chimeric mutants that are 90% S. cerevisiae. Activity was assessed by rescue of Δhem H, a ferrochelatase deficient strain of Escherichia coli, and by enzyme assays. UV-visible and EPR spectroscopy were used to investigate the presence or absence of the [2Fe-2S] cluster. Only 2 of the 13 chimeric mutants that were constructed produced active enzymes. HYB, which is predominately human with the last 40 amino acids being that of S. cerevisiae, is an active protein which does not contain a [2Fe-2S] cluster. The other active chimeric mutant, HSp, is predominately human ferrochelatase with the last 38 amino acids being that of Sc. pombe ferrochelatase. This active mutant contains a [2Fe-2S] cluster, as verified by UV-visible and EPR spectroscopic techniques. No other chimeric proteins had detectable enzyme activity or a [2Fe-2S] cluster. The data are discussed in terms of structural requirements for cluster stability and the role that the cluster plays for ferrochelatase.

Original languageEnglish (US)
Pages (from-to)7461-7467
Number of pages7
JournalBiochemistry
Volume39
Issue number25
DOIs
StatePublished - Jun 27 2000

Fingerprint

Ferrochelatase
Yeast
Yeasts
Schizosaccharomyces
Saccharomyces cerevisiae
Enzyme activity
Enzymes
Amino Acids
Paramagnetic resonance
Ligands
Mutagenesis
Enzyme Assays
Site-Directed Mutagenesis
Escherichia coli
Cysteine
Assays
Spectrum Analysis
Proteins
Iron
Spectroscopy

ASJC Scopus subject areas

  • Biochemistry

Cite this

Examination of the activity of carboxyl-terminal chimeric constructs of human and yeast ferrochelatases. / Medlock, Amy Elizabeth; Dailey, Harry A.

In: Biochemistry, Vol. 39, No. 25, 27.06.2000, p. 7461-7467.

Research output: Contribution to journalArticle

Medlock, Amy Elizabeth ; Dailey, Harry A. / Examination of the activity of carboxyl-terminal chimeric constructs of human and yeast ferrochelatases. In: Biochemistry. 2000 ; Vol. 39, No. 25. pp. 7461-7467.
@article{043baa664f4e47f0a821ef7345016b38,
title = "Examination of the activity of carboxyl-terminal chimeric constructs of human and yeast ferrochelatases",
abstract = "Insertion of ferrous iron into protoporphyrin IX is catalyzed by ferrochelatase (EC 4.99.1.1). Human and Schizosaccharomyces pombe forms of ferrochelatase contain a [2Fe-2S] cluster with three of the four coordinating cysteine ligands located within the 30 carboxyl-terminal residues. Saccharomyces cerevisiae ferrochelatase contains no cluster, but has comparable activity. Truncation mutants of S. cerevisiae lacking either the last 37 or 16 amino acids have no enzyme activity. Chimeric mutants of human, S. cerevisiae, and Sc. pombe ferrochelatase have been created by switching the terminal 10{\%} of the carboxy end of the enzyme. Site-directed mutagenesis has been used to introduce the fourth cysteinyl ligand into chimeric mutants that are 90{\%} S. cerevisiae. Activity was assessed by rescue of Δhem H, a ferrochelatase deficient strain of Escherichia coli, and by enzyme assays. UV-visible and EPR spectroscopy were used to investigate the presence or absence of the [2Fe-2S] cluster. Only 2 of the 13 chimeric mutants that were constructed produced active enzymes. HYB, which is predominately human with the last 40 amino acids being that of S. cerevisiae, is an active protein which does not contain a [2Fe-2S] cluster. The other active chimeric mutant, HSp, is predominately human ferrochelatase with the last 38 amino acids being that of Sc. pombe ferrochelatase. This active mutant contains a [2Fe-2S] cluster, as verified by UV-visible and EPR spectroscopic techniques. No other chimeric proteins had detectable enzyme activity or a [2Fe-2S] cluster. The data are discussed in terms of structural requirements for cluster stability and the role that the cluster plays for ferrochelatase.",
author = "Medlock, {Amy Elizabeth} and Dailey, {Harry A.}",
year = "2000",
month = "6",
day = "27",
doi = "10.1021/bi000134p",
language = "English (US)",
volume = "39",
pages = "7461--7467",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "25",

}

TY - JOUR

T1 - Examination of the activity of carboxyl-terminal chimeric constructs of human and yeast ferrochelatases

AU - Medlock, Amy Elizabeth

AU - Dailey, Harry A.

PY - 2000/6/27

Y1 - 2000/6/27

N2 - Insertion of ferrous iron into protoporphyrin IX is catalyzed by ferrochelatase (EC 4.99.1.1). Human and Schizosaccharomyces pombe forms of ferrochelatase contain a [2Fe-2S] cluster with three of the four coordinating cysteine ligands located within the 30 carboxyl-terminal residues. Saccharomyces cerevisiae ferrochelatase contains no cluster, but has comparable activity. Truncation mutants of S. cerevisiae lacking either the last 37 or 16 amino acids have no enzyme activity. Chimeric mutants of human, S. cerevisiae, and Sc. pombe ferrochelatase have been created by switching the terminal 10% of the carboxy end of the enzyme. Site-directed mutagenesis has been used to introduce the fourth cysteinyl ligand into chimeric mutants that are 90% S. cerevisiae. Activity was assessed by rescue of Δhem H, a ferrochelatase deficient strain of Escherichia coli, and by enzyme assays. UV-visible and EPR spectroscopy were used to investigate the presence or absence of the [2Fe-2S] cluster. Only 2 of the 13 chimeric mutants that were constructed produced active enzymes. HYB, which is predominately human with the last 40 amino acids being that of S. cerevisiae, is an active protein which does not contain a [2Fe-2S] cluster. The other active chimeric mutant, HSp, is predominately human ferrochelatase with the last 38 amino acids being that of Sc. pombe ferrochelatase. This active mutant contains a [2Fe-2S] cluster, as verified by UV-visible and EPR spectroscopic techniques. No other chimeric proteins had detectable enzyme activity or a [2Fe-2S] cluster. The data are discussed in terms of structural requirements for cluster stability and the role that the cluster plays for ferrochelatase.

AB - Insertion of ferrous iron into protoporphyrin IX is catalyzed by ferrochelatase (EC 4.99.1.1). Human and Schizosaccharomyces pombe forms of ferrochelatase contain a [2Fe-2S] cluster with three of the four coordinating cysteine ligands located within the 30 carboxyl-terminal residues. Saccharomyces cerevisiae ferrochelatase contains no cluster, but has comparable activity. Truncation mutants of S. cerevisiae lacking either the last 37 or 16 amino acids have no enzyme activity. Chimeric mutants of human, S. cerevisiae, and Sc. pombe ferrochelatase have been created by switching the terminal 10% of the carboxy end of the enzyme. Site-directed mutagenesis has been used to introduce the fourth cysteinyl ligand into chimeric mutants that are 90% S. cerevisiae. Activity was assessed by rescue of Δhem H, a ferrochelatase deficient strain of Escherichia coli, and by enzyme assays. UV-visible and EPR spectroscopy were used to investigate the presence or absence of the [2Fe-2S] cluster. Only 2 of the 13 chimeric mutants that were constructed produced active enzymes. HYB, which is predominately human with the last 40 amino acids being that of S. cerevisiae, is an active protein which does not contain a [2Fe-2S] cluster. The other active chimeric mutant, HSp, is predominately human ferrochelatase with the last 38 amino acids being that of Sc. pombe ferrochelatase. This active mutant contains a [2Fe-2S] cluster, as verified by UV-visible and EPR spectroscopic techniques. No other chimeric proteins had detectable enzyme activity or a [2Fe-2S] cluster. The data are discussed in terms of structural requirements for cluster stability and the role that the cluster plays for ferrochelatase.

UR - http://www.scopus.com/inward/record.url?scp=0034720740&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0034720740&partnerID=8YFLogxK

U2 - 10.1021/bi000134p

DO - 10.1021/bi000134p

M3 - Article

C2 - 10858295

AN - SCOPUS:0034720740

VL - 39

SP - 7461

EP - 7467

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 25

ER -