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Expression, purification and characterisation of a Bacillus subtilis ferredoxin: a potential electron transfer donor to cytochrome P450 BioI

Green, AJ; Munro, AW; Cheesman, MR; Reid, GA; von Wachenfeldt, Claes LU and Chapman, SK (2003) In Journal of Inorganic Biochemistry 93(1-2). p.92-99
Abstract
The fer gene from Bacillus subtilis has been subcloned and overexpressed in Escherichia coli and the protein (Fer) purified to homogeneity. N-Terminal sequencing and mass spectrometry indicate that the initiator methionine is removed from the protein and that the molecular mass is 8732 Da consistent with that deduced from the gene sequence. Amino-acid sequence comparisons indicate that Fer is a ferredoxin containing a 4Fe-4S cluster. The electron paramagnetic resonance spectrum of the reduced form of Fer is typical for a [4Fe-4S](+) cluster showing rhombic signals with g values of 2.07, 1.93 and 1.88. Reduced Fer also gives rise to a magnetic circular dichroism spectrum typical of a [4Fe-4S] + cluster. Potentiometric titrations indicate... (More)
The fer gene from Bacillus subtilis has been subcloned and overexpressed in Escherichia coli and the protein (Fer) purified to homogeneity. N-Terminal sequencing and mass spectrometry indicate that the initiator methionine is removed from the protein and that the molecular mass is 8732 Da consistent with that deduced from the gene sequence. Amino-acid sequence comparisons indicate that Fer is a ferredoxin containing a 4Fe-4S cluster. The electron paramagnetic resonance spectrum of the reduced form of Fer is typical for a [4Fe-4S](+) cluster showing rhombic signals with g values of 2.07, 1.93 and 1.88. Reduced Fer also gives rise to a magnetic circular dichroism spectrum typical of a [4Fe-4S] + cluster. Potentiometric titrations indicate that Fer has a reduction potential of -385 +/- 10 MV for the [4Fe-4S](+) -[4Fe-4S](2+) redox couple, well within the normal range expected for such a ferredoxin. A proposed physiological role for Fer is as an electron donor to cytochrome P450 BioI. Studies on Fer binding to P450 BioI give rise to a K-d value of 0.87 +/- 0.10 muM. Anaerobic experiments using CO-saturated buffer indicate that Fer is indeed capable of transferring electrons to this cytochrome P450 albeit at a fairly low rate. (C) 2002 Elsevier Science Inc. All rights reserved. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Bacillus subtilis, electron transfer, cytochrome P450, iron-sulfur cluster, ferredoxin, iron-sulfur protein
in
Journal of Inorganic Biochemistry
volume
93
issue
1-2
pages
92 - 99
publisher
Elsevier
external identifiers
  • pmid:12538057
  • wos:000180724800017
  • scopus:0037245378
ISSN
1873-3344
DOI
10.1016/S0162-0134(02)00456-7
language
English
LU publication?
yes
id
12fad873-9145-4a5e-961e-cd2785c516cb (old id 318586)
date added to LUP
2007-09-03 11:58:00
date last changed
2018-09-09 04:18:15
@article{12fad873-9145-4a5e-961e-cd2785c516cb,
  abstract     = {The fer gene from Bacillus subtilis has been subcloned and overexpressed in Escherichia coli and the protein (Fer) purified to homogeneity. N-Terminal sequencing and mass spectrometry indicate that the initiator methionine is removed from the protein and that the molecular mass is 8732 Da consistent with that deduced from the gene sequence. Amino-acid sequence comparisons indicate that Fer is a ferredoxin containing a 4Fe-4S cluster. The electron paramagnetic resonance spectrum of the reduced form of Fer is typical for a [4Fe-4S](+) cluster showing rhombic signals with g values of 2.07, 1.93 and 1.88. Reduced Fer also gives rise to a magnetic circular dichroism spectrum typical of a [4Fe-4S] + cluster. Potentiometric titrations indicate that Fer has a reduction potential of -385 +/- 10 MV for the [4Fe-4S](+) -[4Fe-4S](2+) redox couple, well within the normal range expected for such a ferredoxin. A proposed physiological role for Fer is as an electron donor to cytochrome P450 BioI. Studies on Fer binding to P450 BioI give rise to a K-d value of 0.87 +/- 0.10 muM. Anaerobic experiments using CO-saturated buffer indicate that Fer is indeed capable of transferring electrons to this cytochrome P450 albeit at a fairly low rate. (C) 2002 Elsevier Science Inc. All rights reserved.},
  author       = {Green, AJ and Munro, AW and Cheesman, MR and Reid, GA and von Wachenfeldt, Claes and Chapman, SK},
  issn         = {1873-3344},
  keyword      = {Bacillus subtilis,electron transfer,cytochrome P450,iron-sulfur cluster,ferredoxin,iron-sulfur protein},
  language     = {eng},
  number       = {1-2},
  pages        = {92--99},
  publisher    = {Elsevier},
  series       = {Journal of Inorganic Biochemistry},
  title        = {Expression, purification and characterisation of a Bacillus subtilis ferredoxin: a potential electron transfer donor to cytochrome P450 BioI},
  url          = {http://dx.doi.org/10.1016/S0162-0134(02)00456-7},
  volume       = {93},
  year         = {2003},
}