Possible role of a short extra loop of the long-chain flavodoxin from Azotobacter chroococcum in electron transfer to nitrogenase: Complete H-1, N-15 and C-13 backbone assignments and secondary solution structure of the flavodoxin

The H-1, N-15 and C-13 backbone and H-1 and C-13 beta resonance assignments of the long-chain flavodoxin from Azotobacter chroococcum (the 20-kDa nifF product, flavodoxin-2) in its oxidized form were made at pH 6.5 and 30 degrees C using heteronuclear multidimensional NMR spectroscopy. Analysis of the NOE connectivities, together with amide exchange rates, (3)J(HNH alpha) coupling constants and secondary chemical shifts, provided extensive solution secondary structure information. The secondary structure consists of a five-stranded parallel beta-sheet and five alpha-helices. One of the outer regions of the beta-sheet shows no regular extended conformation, whereas the outer strand beta 4/6 is interrupted by a loop, which is typically observed in long-chain flavodoxins. Two of the five alpha-helices are nonregular at the N-terminus of the helix. Loop regions close to the FMN are identified. Negatively charged amino acid residues are found to be mainly clustered around the FMN, whereas a cluster of positively charged residues is located in one of the alpha-helices. Titration of the flavodoxin with the Fe protein of the A. chroococcum nitrogenase enzyme complex revealed that residues Asn(11), Ser(68) and Asn(72) are involved in complex formation between the flavodoxin and Fe protein. The interaction between the flavodoxin and the Fe protein is influenced by MgADP and is of electrostatic nature.