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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
- Geoghegan, Susan M., Mayhew, Stephen G., Yalloway, Gary N., Butler, Geraldine
- European journal of biochemistry 2000 v.267 no.14 pp. 4434-4444
- Escherichia coli, Megasphaera elsdenii, absorbance, electron transfer, gene expression, genes, glutamic acid, hydroquinone, methionine, mutants, pH, polymerase chain reaction, recombinant proteins, redox potential
- The gene for the electronâtransfer protein flavodoxin has been cloned from Megasphaera elsdenii using the polymerase chain reaction. The recombinant gene was sequenced, expressed in an Escherichia coli expression system, and the recombinant protein purified and characterized. With the exception of an additional methionine residue at the Nâterminus, the physicoâchemical properties of the protein, including its optical spectrum and oxidationâreduction properties, are very similar to those of native flavodoxin. A siteâdirected mutant, E60Q, was made to investigate the effects of removing the negatively charged group that is nearest to N(1) of the bound FMN. The absorbance maximum in the visible region of the bound flavin moves from 446 to 453ânm. The midpoint oxidationâreduction potential at pHâ7 for reduction of oxidized flavodoxin to the semiquinone E2 becomes more negative, decreasing from â114 to â242âmV; E1, the potential for reduction of semiquinone to the hydroquinone, becomes less negative, increasing from â373âmV to â271âmV. A redoxâlinked pKa associated with the hydroquinone is decreased from 5.8 to â¤â4.3. The spectra of the hydroquinones of wildâtype and mutant proteins depend on pH (apparent pKa values of 5.8 and â¤â5.2, respectively). The complexes of apoprotein and all three redox forms of FMN are much weaker for the mutant, with the greatest effect occurring when the flavin is in the semiquinone form. These results suggest that glutamate 60 plays a major role in control of the redox properties of M.âelsdenii flavodoxin, and they provide experimental support to an earlier proposal that the carboxylate on its sideâchain is associated with the redoxâlinked pKa of 5.8 in the hydroquinone.