Structure and Oxidation-Reduction Behavior of 1-Deaza-FMN Flavodoxins: Modulation of Redox Potentials in Flavodoxins

Martha L. Ludwig, Lawrence M. Schopfer, Anita L. Metzger, Katherine A. Pattridge, Vincent Massey

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Abstract

Flavodoxins from Clostridium beijerinckii and from Megasphaera elsdenii with 1-carba-1-deaza-FMN substituted for FMN have been used to study flavin-protein interactions in flavodoxins. The oxidized 1-deaza analogue of FMN binds to apoflavodoxins from M. elsdenii and C. beijerinckii (a.k.a. Clostridium MP) with association constants (Ka) of 1.0 × 10−7 M−1 and 3.1 × 106 M−1, values about 102 less than the corresponding Ka values for FMN. X-ray structure analysis of oxidized 1-deaza-FMN flavodoxin from C. beijerinckii at 2.5-Å resolution shows that the analogue binds with the flavin atoms in the same locations as their equivalents in FMN but that the protein moves in the vicinity of Gly 89 to accommodate the 1-CH group, undergoing displacements which increase the distance between position 1 of the flavin ring and the main-chain atoms of Gly 89 and move the peptide hydrogen of Gly 89 by about 0.6 Å. The X-ray analysis implies that protonation of normal flavin at N(1), as would occur in formation of the neutral fully reduced species, would result in a similar structural perturbation. The oxidation-reduction potentials of 1-deaza-FMN flavodoxin from M. elsdenii have been determined in the pH range 4.5-9.2. The oxidized/semiquinone equilibrium (E′0 = −160 mV at pH 7.0) displays a pH dependence of −60 mV per pH unit; the semiquinone/reduced equilibrium (E′0 = −400 mV at pH 7.0) displays a pH dependence of −60 mV per pH unit at low pH and is pH independent at high pH, with a redox-linked pK of 7.4. Spectral changes of fully reduced 1-deaza-FMN flavodoxin with pH suggest that this latter pK corresponds to protonation of the flavin ring system (the pK of free reduced 1-deaza-FMN is 5.6 [Spencer, R., Fisher, J., & Walsh, C. (1977) Biochemistry 16, 3586-3593]. The pK of reduced 1-deaza-FMN flavodoxin provides an estimate of the electrostatic interaction between the protein and the bound prosthetic group; the free energy of binding neutral reduced 1-deaza-FMN is more negative than that for binding the anionic reduced 1-deaza-FMN by 2.4 kcal. In contrast, the redox-linked pK of native M. elsdenii flavodoxin, which is 5.8, is shown not to be associated with changes in the flavin absorbance, indicating that in the normal FMN flavodoxin structure, the pK determined in studies of the semiquinone/reduced equilibrium must be assigned to the protein rather than to FMN. These data are in agreement with the conclusion from NMR studies [Franken, H.-D., Rüterjans, H., & Muller, F. (1984) Eur. J. Biochem. 138, 481-489] that flavodoxins bind reduced FMN as the anionic species and suggest a mechanism in which unfavorable electrostatic interactions between the negatively charged ring and its protein neighbors play a major role in determining the redox potential of the semiquinone/reduced flavodoxin couple.

Original languageEnglish (US)
Pages (from-to)10364-10375
Number of pages12
JournalBiochemistry
Volume29
Issue number45
DOIs
StatePublished - Nov 1 1990

ASJC Scopus subject areas

  • Biochemistry

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