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 hemo flavoprotein composed of eight alpha-subunits. (SiR-FP) and four beta-subun its (SiR-HP) that catalyses the six electron reduction of sulfite to sulfid e. Each beta-subunit contains a Fe4S4 cluster and a siroheme, and each alph a-subunit binds one FAD and one FMN as prosthetic groups. The FAD gets elec trons 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 Angstrom X-ray structure of SiR-FP60, a recombinant monomeric fragment of SiR-FP th at binds both FAD and FMN and retains the catalytic properties of the nativ e protein. The structure can be divided into three domains. The carboxy-ter minal part of the enzyme is composed of an antiparallel beta-barrel which b inds 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 st ructure of the cytochrome P450 reductase, the third domain, composed of sev en alpha-helices, is supposed to connect the FNR-like module to the N-termi nal flavodoxine-like module. Ln four different crystal forms, the FMN-bindi ng module is absent from electron density maps, although mass spectroscopy, amino acid sequencing and activity experiments carried out on dissolved cr ystals indicate that a functional module is present in the protein. Our res ults clearly indicate that the interaction between the FNR-lke and the FMN- like modules displays lower affinity than in the case of cytochrome P450 re ductase. The flexibility of the FMN-binding domain may be related, as obser ved 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. (C), 2000 Academ ic Press.