Human ferredoxin: Overproduction in Escherichia coli, reconstitution in vitro, and spectroscopic studies of iron-sulfur cluster ligand cysteine-to- serine mutants

Bin Xia, Hong Cheng, Vahe Bandarian, George H. Reed, John L. Markley

Research output: Contribution to journalArticle

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Abstract

Human ferredoxin, the human equivalent of bovine adrenodoxin, is a small iron-sulfur protein with one [2Fe-2S] cluster. It functions, as do other vertebrate ferredoxins, to transfer electrons during the processes of steroid hormone synthesis. A DNA fragment encoding the mature form of human ferredoxin was cloned into an expression vector under control of the T7 RNA polymerase/promoter system. The protein was overproduced in Escherichia coli, and the [2Fe-2S] cluster was incorporated into the protein by in vitro reconstitution. The overall yield was ~30 mg of purified, reconstituted ferredoxin per liter of culture. Four of the five cysteines in human ferredoxin are coordinated to the iron-sulfur cluster. First, the non-ligand cysteine (cysteine-95) was mutated to alanine, and then double mutants were created in which each of the other four cysteines (at positions 46, 52, 55, and 92) were mutated individually to serine. The wild-type ferredoxin and each of the five mutant proteins were studied by UV-visible spectroscopy and electron paramagnetic resonance spectroscopy. The EPR g(av), values of all five mutants were very similar to that of wild-type human ferredoxin. In the reduced state, three of the cysteine-to-serine mutants exhibited axial EPR spectra similar to that of wild-type, but one of the double mutants (C52S/C95A) exhibited a rhombic EPR spectrum. The UV-visible spectroscopic properties of the wild-type and the C95A mutant ferredoxins were identical, but those of the other cysteine-to-serine mutant proteins of human ferredoxin were quite different from those of the wild-type protein and each other. These results, along with those from cysteine-to-serine mutations in other ferredoxins, provide the basis for a more comprehensive theoretical and practical understanding of the features important to the ligation of [2Fe- 2S] clusters, although they do not yet permit determination of which two cysteines ligate Fe(II) and which ligate Fe(III) in the reduced protein.

Original languageEnglish (US)
Pages (from-to)9488-9495
Number of pages8
JournalBiochemistry
Volume35
Issue number29
DOIs
StatePublished - 1996
Externally publishedYes

Fingerprint

Ferredoxins
Sulfur
Serine
Escherichia coli
Cysteine
Iron
Ligands
Paramagnetic resonance
Mutant Proteins
Spectrum Analysis
Proteins
Adrenodoxin
Steroid hormones
In Vitro Techniques
Spectroscopy
Iron-Sulfur Proteins
Electron Spin Resonance Spectroscopy
Alanine
Ligation
Vertebrates

ASJC Scopus subject areas

  • Biochemistry

Cite this

Human ferredoxin : Overproduction in Escherichia coli, reconstitution in vitro, and spectroscopic studies of iron-sulfur cluster ligand cysteine-to- serine mutants. / Xia, Bin; Cheng, Hong; Bandarian, Vahe; Reed, George H.; Markley, John L.

In: Biochemistry, Vol. 35, No. 29, 1996, p. 9488-9495.

Research output: Contribution to journalArticle

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abstract = "Human ferredoxin, the human equivalent of bovine adrenodoxin, is a small iron-sulfur protein with one [2Fe-2S] cluster. It functions, as do other vertebrate ferredoxins, to transfer electrons during the processes of steroid hormone synthesis. A DNA fragment encoding the mature form of human ferredoxin was cloned into an expression vector under control of the T7 RNA polymerase/promoter system. The protein was overproduced in Escherichia coli, and the [2Fe-2S] cluster was incorporated into the protein by in vitro reconstitution. The overall yield was ~30 mg of purified, reconstituted ferredoxin per liter of culture. Four of the five cysteines in human ferredoxin are coordinated to the iron-sulfur cluster. First, the non-ligand cysteine (cysteine-95) was mutated to alanine, and then double mutants were created in which each of the other four cysteines (at positions 46, 52, 55, and 92) were mutated individually to serine. The wild-type ferredoxin and each of the five mutant proteins were studied by UV-visible spectroscopy and electron paramagnetic resonance spectroscopy. The EPR g(av), values of all five mutants were very similar to that of wild-type human ferredoxin. In the reduced state, three of the cysteine-to-serine mutants exhibited axial EPR spectra similar to that of wild-type, but one of the double mutants (C52S/C95A) exhibited a rhombic EPR spectrum. The UV-visible spectroscopic properties of the wild-type and the C95A mutant ferredoxins were identical, but those of the other cysteine-to-serine mutant proteins of human ferredoxin were quite different from those of the wild-type protein and each other. These results, along with those from cysteine-to-serine mutations in other ferredoxins, provide the basis for a more comprehensive theoretical and practical understanding of the features important to the ligation of [2Fe- 2S] clusters, although they do not yet permit determination of which two cysteines ligate Fe(II) and which ligate Fe(III) in the reduced protein.",
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