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

F Talfournier, A W Munro, J Basran, M J Sutcliffe, S Daff, S K Chapman, N S Scrutton

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Abstract

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 languageEnglish
Pages (from-to)20190-20196
Number of pages7
JournalJournal of Biological Chemistry
Volume276
Issue number23
Publication statusPublished - 8 Jun 2001

Keywords

  • CLOSTRIDIUM-BEIJERINCKII FLAVODOXIN
  • FLAVIN MONONUCLEOTIDE COFACTOR
  • TRIMETHYLAMINE DEHYDROGENASE
  • OXIDATION-REDUCTION
  • PARACOCCUS-DENITRIFICANS
  • REDOX POTENTIALS
  • PIG-LIVER
  • PURIFICATION
  • MODULATION
  • GENES

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