Abstract / Description of output
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 <alpha>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.
Original language | English |
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Pages (from-to) | 20190-20196 |
Number of pages | 7 |
Journal | Journal of Biological Chemistry |
Volume | 276 |
Issue number | 23 |
Publication status | Published - 8 Jun 2001 |
Keywords / Materials (for Non-textual outputs)
- CLOSTRIDIUM-BEIJERINCKII FLAVODOXIN
- FLAVIN MONONUCLEOTIDE COFACTOR
- TRIMETHYLAMINE DEHYDROGENASE
- OXIDATION-REDUCTION
- PARACOCCUS-DENITRIFICANS
- REDOX POTENTIALS
- PIG-LIVER
- PURIFICATION
- MODULATION
- GENES