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
Sm. Geoghegan et al., 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, EUR J BIOCH, 267(14), 2000, pp. 4434-4444
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.