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 ERR-m onitored titrations of flavodoxin II of Azotobacter vinelandii (ATCC 4 78). Since wild-type flavodoxin dimerises via intermolecular disulphid e bond formation between Cys69 residues, Cys69 has been replaced by bo th an alanine and a serine residue. Redox properties of the C69A and C 69S flavodoxin mutants were compared to those of wild-type flavodoxin. In the presence of the promotor neomycin, C69A and C69S flavodoxin sh owed a reversible response of the semiquinone/hydroquinone couple at t he glassy carbon electrode. However, the addition of dithiothreitol pr oved to be necessary for the stabilisation of the wild-type flavodoxin response. EPR-monitored redox titrations of wild-type and C69A flavod oxin 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 pK. Instead, the presence of at least two redox-linked protonat ion sites is suggested: pK(red,1) = 5.39 +/- 0.08, pK(ox) = 7.29 +/- 0 .14, and pK(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 o f the semiquinone/hydroquinone redox potential on temperature is -0.5 +/- 0.1 mV . K-1, yielding Delta H degrees = 28.6 +/- 1.5 kJ . mol(-1) and Delta S degrees = -50.0 +/- 6.2 J . mol(-1). K-1. No significant differences in redox properties of wild-type, C69A, and C69S flavodoxi n were observed. The electrochemical data suggest that replacement of Cys69 in the vicinity of the FMN by either an alanine or a serine resi due does not alter the dielectric properties and structure of A. vinel andii flavodoxin II.