Flavodoxin II from Azotobacter vinelandii is a ''long-chain'' flavodoxin and has one of the lowest E 1 midpoint potentials found within the flavodoxin family. To better understand the relationship between structural features and redox potentials, the oxidized form of the C69A mutant of this flavodoxin was crystallized and its three-dimensional structure determined to a resolution of 2.25 Å by molecular replacement. Its overall fold is similar to that of other flavodoxins, with a central five-stranded parallel b-sheet flanked on either side by a-helices. An eight-residue insertion, compared with other long-chain flavodoxins, forms a short 3 10 helix preceding the start of the a 3 helix. The flavin mononucleotide (FMN) cofactor is flanked by a leucine on its re face instead of the more conserved tryptophan, resulting in a more solvent-accessible FMN binding site and stabilization of the hydroquinone (hq) state. In particular the absence of a hydrogen bond to the N5 atom of the oxidized FMN was identified, which destabilizes the ox form, as well as an exceptionally large patch of acidic residues in the vicinity of the FMN N1 atom, which destabilizes the hq form. It is also argued that the presence of a Gly at position 58 in the sequence stabilizes the semiquinone (sq) form, as a result, raising the E 2 value in particular.Keywords: flavodoxin; FMN; hydrogen bonding; polarity; redox potentialsThe redox potential of an electron transfer protein is of prime importance in relation to its function, where the correlation between protein structure and redox potential helps explain how nature has adapted proteins to their specific functions. In the past, this has been studied for a range of flavodoxins, small (14-23 kDa) acidic a/b proteins that contain a single noncovalently bound flavin mononucleotide (FMN) cofactor, from different organisms. In vivo they act as remarkably versatile low potential one-electron donors in a range of reactions (Mayhew and Tollin 1992) in mainly prokaryotic organisms, including obligate and facultative anaerobes, microaerophiles, and photosynthetic cyanobacteria, as well as in both red and green eukaryotic algae. In the photosynthetic bac-

The blue copper protein azurin from Pseudomonas aeruginosa has been covalently labelled with the fluorescing dye Cy5. The optical spectrum of the azurin changes markedly with its redox state. These changes are reflected in the fluorescence intensity of the dye through fluorescence resonance energy transfer (FRET). This provides a sensitive way to monitor biological redox events. The method shown to work in the nanomolar range of protein concentrations, can be easily extended into the sub-nanomolar regime and holds promise for single-molecule detection.

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The reactivity of a variant of the blue copper protein, azurin from Pseudomonas aeruginosa, was investigated with laser flash photolysis and compared with the reactivity of the wild‐type (WT) protein. The variant was obtained by changing the Cu ligating His117 for a glycine. The mutation creates a gap in the ligand shell of the Cu that can be filled with external ligands or water molecules. The crystal structure of the H117G variant is reported. It shows that the immediate surrounding of the Cu site in the variant exhibits less rigidity than in the WT protein and that the loop containing the Cu ligands Cys112, His117 and Met121 in the WT protein has gained flexibility in the H117G variant. Flash photolysis experiments were performed with 5‐deazariboflavin and 8α‐imidazolyl‐(N‐propylyl)‐amino riboflavin as electron donors to probe the reactivity of WT and H117G azurin, and of H117G azurin for which the gap in the Cu co‐ordination shell was filled with imidazole. 8α‐Imidazolyl‐(N‐propylyl)‐amino riboflavin appears one to two orders less efficient as a photo‐flash reductant than 5‐deazariboflavin. The reactivity of the H117G variant in the absence of external ligands appears to be 2.5‐fold lower than the WT reactivity (second‐order rate constants of 51 ± 2 × 107 m−1·s−1 versus 21 ± 1 × 107 m−1·s−1), whereas the addition of imidazole restores reactivity to above the WT level (71 ± 4 × 107 m−1·s−1). The differences are discussed in terms of structural modifications and changes in reorganizational energy and electronic coupling. Database 
Structural data are available in the Protein Data Bank under the accession number . Structured digital abstract  http://www.uniprot.org/uniprot/P00282 http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407 to http://www.uniprot.org/uniprot/P00282 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0114 http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8099460)

A small contraction concomitant with the relaxation of the protein in ca. 3 ls is observed upon ns-laser excitation at 455 nm of the Cys69Ala (C69A) mutant of flavodoxin II from Azotobacter vinelandii. This constitutes another example of detection of a UV-vis silent transient species through a photocalorimetric technique. The contraction is attributed to reorganization of protein dihedral bonds and of water molecules at relatively long distances from the flavin chromophore, after the protein has received the heat shock from the relaxing photoproduced charge transfer state. This study constitutes a preliminary step towards the understanding of the origin of protein movements taking place upon electron transfer reactions, e.g. between a photoinduced electron donor (or acceptor) and an accepting (or donating) cofactor in a protein.