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

The midpoint reduction potentials of the FAD cofactor wild-type Methylophil us methylotrophus (sp. W3A1) electron-transferring flavoprotein (ETF) and t he 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 incomplet e (E'(2) < -250 mV), because of the presence of both kinetic and thermodyna mic 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 a ddition, reduction to the FAD dihydroquinone in alpha R237A ETF is relative ly facile, indicating that the kinetic block seen in wild-type ETF is subst antially removed in the alpha R237A ETF, Thus, kinetic (as well as thermody namic) considerations are important in populating the redox forms of the pr otein-bound flavin, Additionally, we show that electron transfer from trime thylamine dehydrogenase to alpha R237A ETF is severely compromised, because of impaired assembly of the electron transfer complex.