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The crystallographic structure of an engineered flavodoxin mutant from Desulfovibrio vulgaris has been analysed. Site-directed mutagenesis was used to substitute serine 35 with a cysteine to provide a possible covalent linkage. The crystal structure of the semiquinone form of this mutant is similar to the corresponding oxidation state of the wild-type flavodoxin. Analysis of the structural changes reveals the interaction between N(5)H of the flavin and the carbonyl O atom of Gly61 to be critical for modulation of the electrochemical properties of the protein.

Engineered flavodoxins in which a surface residue has been replaced by an exposed cysteine are useful modules to link multi-domain redox proteins obtained by gene fusion to electrode surfaces. In the present work, the crystal structure of the S35C mutant of Desulfovibrio vulgaris flavodoxin in the oxidized state has been determined and compared with a refined structure of the wild type (wt). The structure of wt flavodoxin (space group P4(3)2(1)2, unit-cell parameters a = 50.52, b = 50.52, c = 138.59 A) at 1.34 A resolution has been refined to R = 0.16 and R(free) = 0.18. The structure of the S35C mutant (space group P4(3)2(1)2, unit-cell parameters a = 50.55, b = 50.55, c = 138.39 A) at 1.44 A resolution has been refined to R = 0.13 and R(free) = 0.16. Data sets were collected with synchrotron radiation at 100 K. In the S35C mutant, the Cys35 thiol group points towards a hydrophobic region, whilst in the wt the Ser35 hydroxyl group points towards a more polar region. The solvent exposure of Cys35 is 43 A(2), of which 8 A(2) is for the sulfur. This is comparable to the exposure of 48 A(2) found for the wt Ser35, where that of the hydroxyl oxygen is also 8 A(2).