title: pH-dependent spectroscopic changes associated with the hydroquinone of FMN in flavodoxins.
abstract: Photoreduction with a 5-deazaflavin as the catalyst was used to convert flavodoxins from Desulfovibrio vulgaris, Megasphaera elsdenii, Anabaena PCC 7119, and Azotobacter vinelandii to their hydroquinone forms. The optical spectra of the fully reduced flavodoxins were found to vary with pH in the pH range of 5.0-8.5. The changes correspond to apparent pKa values of 6.5 and 5.8 for flavodoxins from D. vulgaris and M. elsdenii, respectively, values that are similar to the apparent pKa values reported earlier from the effects of pH on the redox potential for the semiquinone-hydroquinone couples of these two proteins (7 and 5.8, respectively). The changes in the spectra resemble those occurring with the free two-electron-reduced flavin for which the pKa is 6.7, but they are red-shifted compared with those of the free flavin. The optical changes occurring with flavodoxins from D. vulgaris and A. vinelandii flavodoxins are larger than those of free reduced FMN. The absorbance of the free and bound flavin increases in the region of 370-390 nm (Delta epsilon = 1-1.8 mM-1 cm-1) with increases of pH. Qualitatively similar pH-dependent changes occur when FMN in D. vulgaris flavodoxin is replaced by iso-FMN, and in the following mutants of D. vulgaris flavodoxin in which the residues mutated are close to the isoalloxazine of the bound flavin: D95A, D95E, D95A/D127A, W60A, Y98S, W60M/Y98W, S96R, and G61A. The 13C NMR spectrum of reduced D. vulgaris [2,4a-13C2]FMN flavodoxin shows two peaks. The peak due to C(4a) is unaffected by pH, but the peak due to C(2) broadens with decreasing pH; the apparent pKa for the change is 6.2. It is concluded that a decrease in pH induces a change in the electronic structure of the reduced flavin due to a change in the ionization state of the flavin, a change in the polarization of the flavin environment, a change in the hydrogen-bonding network around the flavin, and/or possibly a change in the bend along the N(5)-N(10) axis of the flavin. A change in the ionization state of the flavin is the simplest explanation, with the site of protonation differing from that of free FMNH-. The pH effect is unlikely to result from protonation of D95 or D127, the negatively charged amino acids closest to the flavin of D. vulgaris flavodoxin, because the optical changes observed with alanine mutants at these positions are similar to those occurring with the wild-type protein.
authors: Department of Biochemistry, University College Dublin, Belfield, Dublin 4, Ireland.
authors: G N
authors: GN
authors: Yalloway
authors: S G
authors: SG
authors: Mayhew
authors: J P
authors: JP
authors: Malthouse
authors: M E
authors: ME
authors: Gallagher
authors: G P
authors: GP
authors: Curley
mesh_terms: D001059:Apoproteins / Q000737:chemistry; D016969:Desulfovibrio vulgaris / Q000737:chemistry; D005486:Flavin Mononucleotide / Q000737:chemistry* / Q000378:metabolism; D005418:Flavodoxin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006873:Hydroquinones / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D010084:Oxidation-Reduction
chemical_list: D001059:Apoproteins; D005418:Flavodoxin; D006873:Hydroquinones; C026932:apoflavodoxin; C031583:iso-FMN; D005486:Flavin Mononucleotide; C031927:hydroquinone