Structural and Phylogenetic Analysis of Rhodobacter capsulatus NifF: Uncovering General Features of Nitrogen-fixation (nif)-Flavodoxins

Abstract

Analysis of the crystal structure of NifF from Rhodobacter capsulatus and its homologues reported so far reflects the existence of unique structural features in nif flavodoxins: a leucine at the re face of the isoalloxazine, an eight-residue insertion at the C-terminus of the 50’s loop and a remarkable difference in the electrostatic potential surface with respect to non-nif flavodoxins. A phylogenetic study on 64 sequences from 52 bacterial species revealed four clusters, including different functional prototypes, correlating the previously defined as “short-chain” with the firmicutes flavodoxins and the “long-chain” with gram-negative species. The comparison of Rhodobacter NifF structure with other bacterial flavodoxin prototypes discloses the concurrence of specific features of these functional electron donors to nitrogenase.

The ribosome regulates flavodoxin folding

“…As explained above, the circumstances under which proteins are introduced in in vivo or in vitro experiments (i.e., full-length sequence versus growing polypeptide) differ considerably. Though in some cases chemically or sequentially truncated versions of proteins have been studied for their folding behaviour (21)(22)(23)(24), these experiments do not take into account the other major differences between in vivo and in vitro circumstances. As the lifetimes of proteins in cells can range from minutes to years (25) there is ample time for multiple folding rounds for fully synthesized protein.…”

“…If they predominantly locate in the N-terminal part of multi-domain proteins this protection against degradation and aggregation may be an important factor during cotranslational folding of these proteins. One such example is the group of P450 fusion proteins, in which the N-terminal domain is in all but one case either a heme-binding P450 or a flavin-binding domain (23).…”

“…In contrast, off-pathway MGs contain non-native secondary structure and/or packing. Nowadays, many proteins are thought to fold via MGs (17)(18)(19)(20)(21)(22)(23) and these intermediates are for example necessary during insertion of proteins into membranes or during translocation (24)(25)(26). Because of their importance in protein folding, considerable effort has been put into determining the structural properties of MGs.…”

“…Heme has been suggested to bind to nascent globin (7), which may imply that the protein folds cotranslationally. Flavin adenine dinucleotide (FAD) was proposed to covalently attach to nascent 6-hydroxy-D-nicotine oxidase (22), but its cotranslational flavinylation is now judged unlikely (23).…”

“…Such insertions often happen cotranslationally, i.e., while the ribosome synthesizes the protein concerned. It has been shown that proteins can fold cotranslationally and sample intermediate folding states (17)(18)(19)(20)(21)(22)(23), which might include MGs.…”