Four crystal structures of the 60 kDa flavoprotein monomer of the sulfite reductase indicate a disordered flavodoxin-like module

Escherichia coli NADPH-sulfite reductase (SiR) is a 780 kDa multimeric hemoflavoprotein composed of eight alpha-subunits (SiR-FP) and four beta-subunits (SiR-HP) that catalyses the six electron reduction of sulfite to sulfide. Each beta-subunit contains a Fe4S4 cluster and a siroheme, and each alpha...

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Bibliographic Details
Published in:Journal of molecular biology 2000-05, Vol.299 (1), p.199-212
Main Authors: Gruez, A, Pignol, D, Zeghouf, M, Covès, J, Fontecave, M, Ferrer, J L, Fontecilla-Camps, J C
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Binding Sites
Catalysis
Crystallization
Crystallography, X-Ray
Escherichia coli
Escherichia coli - enzymology
Flavin Mononucleotide - metabolism
Flavin-Adenine Dinucleotide - metabolism
Flavodoxin - chemistry
Flavodoxin - metabolism
Models, Molecular
Molecular Sequence Data
Molecular Weight
Motion
NADH, NADPH Oxidoreductases - chemistry
NADH, NADPH Oxidoreductases - metabolism
NADP - metabolism
NADPH-Ferrihemoprotein Reductase
NADPH-sulfite reductase
Oxidoreductases Acting on Sulfur Group Donors - chemistry
Oxidoreductases Acting on Sulfur Group Donors - metabolism
Peptide Fragments - chemistry
Peptide Fragments - metabolism
Pliability
Protein Binding
Protein Structure, Secondary
Protein Structure, Tertiary
Sequence Alignment
Static Electricity
Sulfite Reductase (NADPH)
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Summary:Escherichia coli NADPH-sulfite reductase (SiR) is a 780 kDa multimeric hemoflavoprotein composed of eight alpha-subunits (SiR-FP) and four beta-subunits (SiR-HP) that catalyses the six electron reduction of sulfite to sulfide. Each beta-subunit contains a Fe4S4 cluster and a siroheme, and each alpha-subunit binds one FAD and one FMN as prosthetic groups. The FAD gets electrons from NADPH, and the FMN transfers the electrons to the metal centers of the beta-subunit for sulfite reduction. We report here the 1.94 A X-ray structure of SiR-FP60, a recombinant monomeric fragment of SiR-FP that binds both FAD and FMN and retains the catalytic properties of the native protein. The structure can be divided into three domains. The carboxy-terminal part of the enzyme is composed of an antiparallel beta-barrel which binds the FAD, and a variant of the classical pyridine dinucleotide binding fold which binds NADPH. These two domains form the canonic FNR-like module, typical of the ferredoxin NADP+ reductase family. By analogy with the structure of the cytochrome P450 reductase, the third domain, composed of seven alpha-helices, is supposed to connect the FNR-like module to the N-terminal flavodoxine-like module. In four different crystal forms, the FMN-binding module is absent from electron density maps, although mass spectroscopy, amino acid sequencing and activity experiments carried out on dissolved crystals indicate that a functional module is present in the protein. Our results clearly indicate that the interaction between the FNR-like and the FMN-like modules displays lower affinity than in the case of cytochrome P450 reductase. The flexibility of the FMN-binding domain may be related, as observed in the case of cytochrome bc1, to a domain reorganisation in the course of electron transfer. Thus, a movement of the FMN-binding domain relative to the rest of the enzyme may be a requirement for its optimal positioning relative to both the FNR-like module and the beta-subunit.
ISSN:0022-2836
1089-8638
DOI:10.1006/jmbi.2000.3748