The Flavoprotein Component of the Escherichia coli Sulfite Reductase: Expression, Purification, and Spectral and Catalytic Properties of a Monomeric Form Containing both the Flavin Adenine Dinucleotide and the Flavin Mononucleotide Cofactors

The flavoprotein component (SiR-FP) of the sulfite reductase from Escherichia coli is an octamer containing one FAD and one FMN per polypeptide chain. SiR-FP60, a SiR-FP fragment starting with alanine-52, was overexpressed in E. coli and purified as a monomer. The N-terminal part of the native prote...

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Bibliographic Details
Published in:Biochemistry (Easton) 1998-04, Vol.37 (17), p.6114-6123
Main Authors: Zeghouf, Mahel, Fontecave, Marc, Macherel, David, Covès, Jacques
Format: Article
Language:English
Subjects:
Catalysis
Dithionite
Electron Spin Resonance Spectroscopy
Endopeptidases
Energy Transfer
Escherichia coli - enzymology
Flavin Mononucleotide - chemistry
Flavin-Adenine Dinucleotide - chemistry
Flavins - chemistry
Flavoproteins - biosynthesis
Flavoproteins - chemistry
Flavoproteins - isolation & purification
Hydrolysis
Molecular Weight
NADP
Oxidation-Reduction
Oxidoreductases Acting on Sulfur Group Donors - biosynthesis
Oxidoreductases Acting on Sulfur Group Donors - chemistry
Oxidoreductases Acting on Sulfur Group Donors - isolation & purification
Peptide Fragments - chemistry
Peptide Fragments - isolation & purification
Recombinant Proteins - biosynthesis
Recombinant Proteins - isolation & purification
Spectrophotometry
Titrimetry
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Summary:The flavoprotein component (SiR-FP) of the sulfite reductase from Escherichia coli is an octamer containing one FAD and one FMN per polypeptide chain. SiR-FP60, a SiR-FP fragment starting with alanine-52, was overexpressed in E. coli and purified as a monomer. The N-terminal part of the native protein contains thus all the determinants required for the polymerization. SiR-FP60 retains both FAD and FMN with comparable contributions of the two flavins and the catalytic properties of SiR-FP. Thus, SiR-FP60 can be considered as a reliable simplified model of the sulfite reductase flavoprotein component. The formation and the stabilization of the neutral FMN semiquinone is thermodynamically favorable in SiR-FP60 upon reduction with photoreduced deazaflavin, dithionite, or NADPH. Generation of FMNH• is explained from a disproportionation of electrons between the reduced and oxidized FMN moieties during an intermolecular reaction, as shown with SiR-FP23, the FMN-binding domain of SiR-FP. The neutral FAD semiquinone can be observed only within SiR-FP43, the isolated FAD-binding domain. NADPH was used as a titrant or in excess to demonstrate that electron transfer is possible only because the FMN cofactor is coupled to FAD as an electron acceptor in the protein. The electron distribution within the various reduced forms of SiR-FP60 has been compared with that of the reduced forms of cytochrome P450 reductase, bacterial cytochrome P450, and nitric-oxide synthase. Despite the conservation of the bi-flavin-domain structure between these proteins over evolutionary time, each of them provides significantly different flavin reactivities.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi9728699