title: alphaT244M mutation affects the redox, kinetic, and in vitro folding properties of Paracoccus denitrificans electron transfer flavoprotein.
abstract: Threonine 244 in the alpha subunit of Paracoccus denitrificans transfer flavoprotein (ETF) lies seven residues to the amino terminus of a proposed dinucleotide binding motif for the ADP moiety of the FAD prosthetic group. This residue is highly conserved in the alpha subunits of all known ETFs, and the most frequent pathogenic mutation in human ETF encodes a methionine substitution at the corresponding position, alphaT266. The X-ray crystal structures of human and P. denitrificans ETFs are very similar. The hydroxyl hydrogen and a backbone amide hydrogen of alphaT266 are hydrogen bonded to N(5) and C(4)O of the flavin, respectively, and the corresponding alphaT244 has the same structural role in P. denitrificans ETF. We substituted a methionine for T244 in the alpha subunit of P. denitrificans ETF and expressed the mutant ETF in Escherichia coli. The mutant protein was purified, characterized, and compared with wild type P. denitrificans ETF. The mutation has no significant effect on the global structure of the protein as inferred from visible and near-ultraviolet absorption and circular dichroism spectra, far-ultraviolet circular dichroism spectra, and infrared spectra in 1H2O and 2H2O. Intrinsic fluorescence due to tryptophan of the mutant protein is 60% greater than that of the wild type ETF. This increased tryptophan fluorescence is probably due to a change in the environment of the nearby W239. Tyrosine fluorescence is unchanged in the mutant protein, although two tyrosine residues are close to the site of the mutation. These results indicate that a change in structure is minor and localized. Kinetic constants of the reductive half-reaction of ETF with porcine medium chain acyl-CoA dehydrogenase are unaltered when alphaT244M ETF serves as the substrate; however, the mutant ETF fails to exhibit saturation kinetics when the semiquinone form of the protein is used as the substrate in the disproportionation reaction catalyzed by P. denitrificans electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO). The redox behavior of the mutant ETF was also altered as determined from the equilibrium constant of the disproportionation reaction. The separation of flavin redox potentials between the oxidized/semiquinone couple and semiquinone/hydroquinone couple are -6 mV in the wild type ETF and -27 mV in the mutant ETF. The mutation does not alter the AMP content of the protein, although the extent and fidelity of AMP-dependent, in vitro renaturation of the mutant AMP-free apoETF is reduced by 57% compared to renaturation of wild type apoETF, likely due to the absence of the potential hydrogen bond donor T244.
authors.affiliation: Department of Pediatrics, University of Colorado School of Medicine, Denver 80262, USA.
authors.forename: K J
authors.initials: KJ
authors.lastname: Griffin
authors.forename: T M
authors.initials: TM
authors.lastname: Dwyer
authors.forename: M C
authors.initials: MC
authors.lastname: Manning
authors.forename: J D
authors.initials: JD
authors.lastname: Meyer
authors.forename: J F
authors.initials: JF
authors.lastname: Carpenter
authors.forename: F E
authors.initials: FE
authors.lastname: Frerman
mesh_terms: D000249:Adenosine Monophosphate / Q000032:analysis; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D044927:Electron-Transferring Flavoproteins; D004926:Escherichia coli / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D005420:Flavoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005609:Free Radicals / Q000378:metabolism; D015870:Gene Expression; D007259:Infrared Rays; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010084:Oxidation-Reduction; D010231:Paracoccus denitrificans / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D014364:Tryptophan / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D044927:Electron-Transferring Flavoproteins; D005420:Flavoproteins; D005609:Free Radicals; D005182:Flavin-Adenine Dinucleotide; D000249:Adenosine Monophosphate; D014364:Tryptophan