Cloning, sequencing and expression of the gene for flavodoxin from Megasphaera elsdenii and the effects of removing the protein negative charge that is closest to N(1) of the bound FMN

The gene for the electron-transfer protein flavodoxin has been cloned from Megasphaera elsdenii using the polymerase chain reaction. The recombinant g ene was sequenced, expressed in an Escherichia coli expression system, and the recombinant protein purified and characterized. With the exception of a n additional methionine residue at the N-terminus, the physico-chemical pro perties of the protein, including its optical spectrum and oxidation-reduct ion properties, are very similar to those of native flavodoxin. A site-dire cted mutant, E60Q, was made to investigate the effects of removing the nega tively charged group that is nearest to N(1) of the bound FMN. The absorban ce maximum in the visible region of the bound flavin moves from 446 to 453 nm. The midpoint oxidation-reduction potential at pH 7 for reduction of oxi dized flavodoxin to the semiquinone E-2 becomes more negative, decreasing f rom -114 to -242 mV; E-1, the potential for reduction of semiquinone to the hydroquinone, becomes less negative, increasing from -373 mV to -271 mV. A redox-linked pK(a) associated with the hydroquinone is decreased from 5.8 to less than or equal to 4.3. The spectra of the hydroquinones of wild-type and mutant proteins depend on pH (apparent pK(a) values of 5.8 and less th an or equal to 5.2, respectively). The complexes of apoprotein and all thre e redox forms of FMN are much weaker for the mutant, with the greatest effe ct occurring when the flavin is in the semiquinone form. These results sugg est that glutamate 60 plays a major role in control of the redox properties of M. elsdenii flavodoxin, and they provide experimental support to an ear lier proposal that the carboxylate on its side-chain is associated with the redox-linked pK(a) of 5.8 in the hydroquinone.