Apoflavodoxin: Structure, stability, and FMN binding

Flavodoxins are one domain alpha/beta electron transfer proteins that parti cipate in photosynthetic reactions. All flavodoxins carry a molecule of fla vin mononucleotide (FMN), non-covalently bound, that confers redox properti es to the protein. There are two structurally distinct flavodoxins, short o nes and long flavodoxins; the latter contain an extra loop with unknown fun ction. We have undertaken the study of the stability and folding of the apo flavodoxin from Anabaena (a long flavodoxin) and the analysis of the intera ction between the apoflavodoxin and FMN. Our studies indicate that apoflavo doxin folds in a few seconds to a form that is competent in FMN binding. Th e stability of this apoflavodoxin is low and its urea denaturation can be d escribed by a two-state mechanism. The role of the different parts of the a poflavodoxin in the stability and structure of the whole protein is being i nvestigated using mutagenesis and specific cleavage to generate apoflavodox in fragments. The X-ray structure of apoflavodoxin is very similar to that of its complex with FMN, the main difference being the conformation of the two aromatic residues that sandwich FMN in the complex. In apoflavodoxin th ese groups interact with each other so closing the FMN binding site. Despit e this fact, apoflavodoxin binds FMN tightly and rapidly, and the resulting holoflavodoxin displays a high conformational stability. We have found tha t one role of the aromatic residues that interact with FMN is to help to re tain bound the reduced form of the cofactor whose complex with apoflavodoxi n is otherwise too weak. (C) Societe francaise de biochimie et biologie mol eculaire / Elsevier, Paris.