Redox Properties of Wild‐Type, Cys69Ala, and Cys69Ser Azotobacter Vinelandii Flavodoxin II as Measured by Cyclic Voltammetry and EPR Spectroscopy

This study deals with the detailed electrochemistry and complete EPR‐monitored titrations of flavodoxin II of Azotobacter vinelandii (ATCC 478). Since wild‐type flavodoxin dimerises via intermolecular disulphide bond formation between Cys69 residues, Cys69 has been replaced by both an alanine and a serine residue. Redox properties of the C69A and C69S flavodoxin mutants were compared to those of wild‐type flavodoxin. In the presence of the promotor neomycin, C69A and C69S flavodoxin showed a reversible response of the semiquinone/hydroquinone couple at the glassy carbon electrode. However, the addition of dithiothreitol proved to be necessary for the stabilisation of the wild‐type flavodoxin response. EPR‐monitored redox titrations of wild‐type and C69A flavodoxin at high and low pH confirmed the redox potentials measured using cyclic voltammetry. The pH dependence of the semiquinone/hydroquinone redox potentials cannot be described using a model assuming one redox‐linked p K. Instead, the presence of at least two redox‐linked protonation sites is suggested: p K red,1 = 5.39±0.08, p K ox = 7.29±0.14, and p K red,2 = 7.84±0.14 with E m,7 =−459±4 mV, and a constant redox potential at high pH of −485±4 mV. The dependence of the semiquinone/hydroquinone redox potential on temperature is −0.5±0.1 mV·K −1 , yielding ?H°= 28.6±1.5 kJ·mol −1 and ?S°= ‐50.0±6.2 J·mol −1 ·K −1 . No significant differences in redox properties of wild‐type, C69A, and C69S flavodoxin were observed. The electrochemical data suggest that replacement of Cys69 in the vicinity of the FMN by either an alanine or a serine residue does not alter the dielectric properties and structure of A. vinelandii flavodoxin II.