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

Amanda J Green, Andrew W Munro, Myles R Cheesman, Graeme A Reid, C von Wachenfeldt, Stephen K Chapman

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    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.

    Original languageEnglish
    Pages (from-to)92-99
    Number of pages8
    JournalJournal of Inorganic Biochemistry
    Volume93
    Issue number1-2
    DOIs
    Publication statusPublished - 1 Jan 2003

    Keywords

    • ESCHERICHIA-COLI
    • iron-sulfur cluster
    • iron-sulfur protein
    • Bacillus subtilis
    • PROTEIN
    • electron transfer
    • ferredoxin
    • cytochrome P450
    • MAGNETIC-CIRCULAR-DICHROISM
    • BACTERIAL FERREDOXINS
    • FLAVODOXIN
    • REDUCTASE
    • IDENTIFICATION
    • 4FE-4S FERREDOXIN

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