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 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 N-terminus, the physico-chemical properties of the protein, including its optical spectrum and oxidation-reduction properties, are very similar to those of native flavodoxin. A site-directed 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 453 nm. The midpoint oxidation-reduction potential at pH 7 for reduction of oxidized flavodoxin to the semiquinone E2 becomes more negative, decreasing from -114 to -242 mV; E1, the potential for reduction of semiquinone to the hydroquinone, becomes less negative, increasing from -373 mV to -271 mV. A redox-linked pKa associated with the hydroquinone is decreased from 5.8 to < or = 4.3. The spectra of the hydroquinones of wild-type and mutant proteins depend on pH (apparent pKa values of 5.8 and < or = 5.2, 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. elsdenii flavodoxin, and they provide experimental support to an earlier proposal that the carboxylate on its side-chain is associated with the redox-linked pKa of 5.8 in the hydroquinone.
Geoghegan
S M
SM
Department of Biochemistry, University College Dublin, Belfield, Ireland.
Mayhew
S G
SG
Yalloway
G N
GN
Butler
G
G
D000595:Amino Acid Sequence; D001406:Bacillaceae / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005418:Flavodoxin / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D018698:Glutamic Acid / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006873:Hydroquinones / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013696:Temperature; D013816:Thermodynamics
D005418:Flavodoxin; D006873:Hydroquinones; D011994:Recombinant Proteins; D018698:Glutamic Acid; C031927:hydroquinone