alpha Arg-237 in Methylophilus methylotrophus (sp. W3A1) electron-transferring flavoprotein affords approximately 200-millivolt stabilization of the FAD anionic semiquinone and a kinetic block on full reduction to the dihydroquinone.

The midpoint reduction potentials of the FAD cofactor in wild-type Methylophilus methylotrophus (sp. W3A1) electron-transferring flavoprotein (ETF) and the alphaR237A 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 alphaArg-237 for Ala in M. methylotrophus ETF is to engineer a remarkable approximately 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 alphaR237A ETF is relatively facile, indicating that the kinetic block seen in wild-type ETF is substantially removed in the alphaR237A 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 alphaR237A ETF is severely compromised, because of impaired assembly of the electron transfer complex.