Modulation of the redox properties of the flavin cofactor through hydrogen-bonding interactions with the N(5) atom: role of alphaSer254 in the electron-transfer flavoprotein from the methylotrophic bacterium W3A1.
The functional effects of hydrogen-bonding interactions at the N(5) atom of the flavin cofactors in the oxidized state have not been well established in flavoproteins. The unique properties of the electron-transfer flavoprotein from the methylotrophic bacteria W3A1 (wETF) were used to advantage in this study to evaluate this interaction. In wETF, the side-chain hydroxyl group of alphaSer254 serves as a hydrogen bond donor to the N(5) atom in the oxidized state of the flavin. The strength of this hydrogen bond was systematically altered by the substitution of alphaSer254 with threonine, cysteine, or alanine by site-directed mutagenesis. The anionic semiquinone form of the flavin, which is highly stabilized both thermodynamically and kinetically in the wild-type protein, was observed to accumulate in all three mutants. However, the midpoint potential for the first couple (Eox/sq) was significantly decreased for all of the mutants, and the kinetic barrier toward the reduction of the anionic semiquinone that is observed in the wild-type wETF was effectively abolished in the alphaS254T and alphaS254C mutants. Based on the observed changes in the Kd values and associated binding energies for the flavin, the amino acid replacements destabilize both the oxidized and semiquinone states of the flavin, but to a much greater extent for the anionic semiquinone state. The Eox/sq values for the alphaSer254 mutants follow a general trend with the strength of N(5) H-bond in the oxidized state as indicated by Raman spectral analyses. These results support the conclusion that the H-bonding interaction at the N(5) plays a key role in establishing the high Eox/sq and the unusually high stability of the anionic semiquinone state in wETF.
Yang
Kun-Yun
KY
Biophysics Program, The Ohio State University, Columbus, Ohio 43210, USA.
Swenson
Richard P
RP
D001483:Base Sequence; D017931:DNA Primers; D019605:Euryarchaeota / Q000737:chemistry*; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry*; D005420:Flavoproteins / Q000737:chemistry* / Q000235:genetics; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D012694:Serine / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet; D013059:Spectrum Analysis, Raman
D017931:DNA Primers; D005420:Flavoproteins; D005182:Flavin-Adenine Dinucleotide; D012694:Serine