TY - JOUR
T1 - alpha Arg-237 in Methylophilus methylotrophus (sp W3A1) electron-transferring flavoprotein affords similar to 200-millivolt stabilization of the FAD anionic semiquinone and a kinetic block on full reduction to the dihydroquinone
AU - Talfournier, François
AU - Munro, Andrew W
AU - Basran, Jaswir
AU - Sutcliffe, Michael J
AU - Daff, Simon N
AU - Chapman, Stephen K
AU - Scrutton, Nigel S
PY - 2001/6/8
Y1 - 2001/6/8
N2 - The midpoint reduction potentials of the FAD cofactor wild-type Methylophilus methylotrophus (sp. W3A1) electron-transferring flavoprotein (ETF) and the alpha R237A mutant were determined by anaerobic redox titration. The FAD reduction potential of the oxidized-semiquinone couple in wild-type ETF (E'(1)) is + 153 +/- 2 mV, indicating exceptional stabilization of the flavin anionic semiquinone species. Conversion to the dihydroquinone is incomplete (E'(2) <-250 mV), because of the presence of both kinetic and thermodynamic blocks on full reduction of the FAD, A structural model of ETF (Chohan, K. K., Scrutton, N. S., and Sutcliffe, M. J. (1998) Protein Pept. Lett. 5, 231-236) suggests that the guanidinium group of Arg-237, which is located over the si face of the flavin isoalloxazine ring, plays a key role in the exceptional stabilization of the anionic semiquinone in wild-type ETF, The major effect of exchanging Arg-237 for Ala in M, methylotrophus ETF is to engineer a remarkable similar to 200-mV destabilization of the flavin anionic semiquinone (E'(2) = -31 +/- 2 mV, and E'(1) = -43 +/- 2 mV). In addition, reduction to the FAD dihydroquinone in alpha R237A ETF is relatively facile, indicating that the kinetic block seen in wild-type ETF is substantially removed in the alpha R237A ETF, Thus, kinetic (as well as thermodynamic) considerations are important in populating the redox forms of the protein-bound flavin, Additionally, we show that electron transfer from trimethylamine dehydrogenase to alpha R237A ETF is severely compromised, because of impaired assembly of the electron transfer complex.
AB - The midpoint reduction potentials of the FAD cofactor wild-type Methylophilus methylotrophus (sp. W3A1) electron-transferring flavoprotein (ETF) and the alpha R237A mutant were determined by anaerobic redox titration. The FAD reduction potential of the oxidized-semiquinone couple in wild-type ETF (E'(1)) is + 153 +/- 2 mV, indicating exceptional stabilization of the flavin anionic semiquinone species. Conversion to the dihydroquinone is incomplete (E'(2) <-250 mV), because of the presence of both kinetic and thermodynamic blocks on full reduction of the FAD, A structural model of ETF (Chohan, K. K., Scrutton, N. S., and Sutcliffe, M. J. (1998) Protein Pept. Lett. 5, 231-236) suggests that the guanidinium group of Arg-237, which is located over the si face of the flavin isoalloxazine ring, plays a key role in the exceptional stabilization of the anionic semiquinone in wild-type ETF, The major effect of exchanging Arg-237 for Ala in M, methylotrophus ETF is to engineer a remarkable similar to 200-mV destabilization of the flavin anionic semiquinone (E'(2) = -31 +/- 2 mV, and E'(1) = -43 +/- 2 mV). In addition, reduction to the FAD dihydroquinone in alpha R237A ETF is relatively facile, indicating that the kinetic block seen in wild-type ETF is substantially removed in the alpha R237A ETF, Thus, kinetic (as well as thermodynamic) considerations are important in populating the redox forms of the protein-bound flavin, Additionally, we show that electron transfer from trimethylamine dehydrogenase to alpha R237A ETF is severely compromised, because of impaired assembly of the electron transfer complex.
KW - GENES
KW - TRIMETHYLAMINE DEHYDROGENASE
KW - FLAVIN MONONUCLEOTIDE COFACTOR
KW - PIG-LIVER
KW - PURIFICATION
KW - REDOX POTENTIALS
KW - CLOSTRIDIUM-BEIJERINCKII FLAVODOXIN
KW - PARACOCCUS-DENITRIFICANS
KW - MODULATION
KW - OXIDATION-REDUCTION
U2 - 10.1074/jbc.M010853200
DO - 10.1074/jbc.M010853200
M3 - Article
SN - 0021-9258
VL - 276
SP - 20190
EP - 20196
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 23
ER -