E. Steensma et al., REDOX PROPERTIES OF WILD-TYPE, CYS69ALA, AND CYS69SER AZOTOBACTER-VINELANDII FLAVODOXIN-II AS MEASURED BY CYCLIC VOLTAMMETRY AND EPR SPECTROSCOPY, European journal of biochemistry, 235(1-2), 1996, pp. 167-172
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e bond formation between Cys69 residues, Cys69 has been replaced by bo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.