title: Redox properties of wild-type, Cys69Ala, and Cys69Ser Azotobacter vinelandii flavodoxin II as measured by cyclic voltammetry and EPR spectroscopy,.
abstract: 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 Since wild-type flavodoxin dimerises via intermolecular disulphide bond formation between Cys69 residues, Cys69 has been replaced by both an alanine and a serine residue.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 pK. Instead, the presence of at least two redox-linked protonation sites is suggested: pKred.1 = 5.39 +/- 0.08, pKox = 7.29 +/- 0.14, and pKred.2 = 7.84 +/- 0.14 with Em.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 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 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.
authors.affiliation: Department of Biochemistry, Wageningen Agricultural University, The Netherlands.
authors.forename: E
authors.initials: E
authors.lastname: Steensma
authors.forename: H A
authors.initials: HA
authors.lastname: Heering
authors.forename: W R
authors.initials: WR
authors.lastname: Hagen
authors.forename: C P
authors.initials: CP
authors.lastname: Van Mierlo
mesh_terms: D016948:Azotobacter vinelandii / Q000235:genetics / Q000378:metabolism*; D004563:Electrochemistry; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics; D005418:Flavodoxin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D015394:Molecular Structure; D010084:Oxidation-Reduction; D017354:Point Mutation
chemical_list: D005418:Flavodoxin