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.
Talfournier
F
F
Departments of Biochemistry and Chemistry, University of Leicester, University Road, Leicester LE1 7RH, United Kingdom.
Munro
A W
AW
Basran
J
J
Sutcliffe
M J
MJ
Daff
S
S
Chapman
S K
SK
Scrutton
N S
NS
D001120:Arginine / Q000378:metabolism*; D001483:Base Sequence; D016227:Benzoquinones / Q000378:metabolism*; D017931:DNA Primers; D044927:Electron-Transferring Flavoproteins; D005420:Flavoproteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D020630:Methylophilus methylotrophus / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011199:Potentiometry; D011809:Quinones / Q000378:metabolism*
C403008:1,4-dihydroquinone; D016227:Benzoquinones; D017931:DNA Primers; D044927:Electron-Transferring Flavoproteins; D005420:Flavoproteins; D011809:Quinones; C025232:semiquinone radicals; D001120:Arginine