pH-Dependent spectroscopic changes associated with the hydroquinone of FMNin flavodoxins

Photoreduction with a 5-deazaflavin as the catalyst was used to convert fla vodoxins from Desulfovibrio vulgaris, Megasphaera elsdenii, Anabaena PCC 71 19, and Azotobacter vinelandii to their hydroquinone forms. The optical spe ctra 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 pK(a) values of 6.5 an d 5.8 for flavodoxins from D. vulgaris and M. elsdenii, respectively, value s that are similar to the apparent pK(a) values reported earlier from the e ffects of pH on the redox potential for the semiquinone-hydroquinone couple s of these two proteins (7 and 5.8, respectively). The changes in the spect ra resemble those occurring with the free two-electron-reduced flavin for w hich the pK(a) is 6.7, but they are red-shifted compared with those of the free flavin. The optical changes occurring with flavodoxins from D. vulgari s 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. Qualitat ively similar pH-dependent changes occur when FMN in D. vulgaris flavodoxin is replaced by iso-FMN, and in the following mutants of D. vulgaris flavod oxin in which the residues mutated are close to the isoalloxazine of the bo und flavin: D95A, D95E, D95A/D127A, W60A, Y98S, W60M/Y98W, S96R, and G61A. The C-13 NMR spectrum of reduced D. vulgaris [2,4a-C-13(2)]FMN flavodoxin s hows 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 pK(a) for the change is 6.2, It is concluded that a decrease in pH induces a change in the electron ic 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 ch ange in the hydrogen-bonding network around the flavin, and/or possibly a c hange 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 o f protonation differing from that of free FMNH-. The pH effect is unlikely to result from protonation of D95 or D127, the negatively charged amino aci ds closest to the flavin of D. vulgaris flavodoxin, because the optical cha nges observed with alanine mutants at these positions are similar to those occurring with the wild-type protein.