Expression, purification and characterisation of a Bacillus subtilis ferredoxin: a potential electron transfer donor to cytochrome P450 BioI.
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 microM. 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.
Green
Amanda J
AJ
Department of Chemistry, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, UK.
Munro
Andrew W
AW
Cheesman
Myles R
MR
Reid
Graeme A
GA
von Wachenfeldt
Claes
C
Chapman
Stephen K
SK
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000737:chemistry* / Q000235:genetics; D002942:Circular Dichroism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D005288:Ferredoxins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D015870:Gene Expression; D008024:Ligands; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011485:Protein Binding; D016415:Sequence Alignment
D005288:Ferredoxins; D008024:Ligands; D003577:Cytochrome P-450 Enzyme System