Differential coupling through Val-344 and Tyr-442 of trimethylamine dehydrogenase in electron transfer reactions with ferricenium ions and electron transferring flavoprotein.
Modeling studies of the trimethylamine dehydrogenase-electron transferring flavoprotein (TMADH-ETF) electron transfer complex have suggested potential roles for Val-344 and Tyr-442, found on the surface of TMADH, in electronic coupling between the 4Fe-4S center of TMADH and the FAD of ETF. The importance of these residues in electron transfer, both to ETF and to the artificial electron acceptor, ferricenium (Fc(+)), has been studied by site-directed mutagenesis and stopped-flow spectroscopy. Reduction of the 6-(S)-cysteinyl FMN in TMADH is not affected by mutation of either Tyr-442 or Val-344 to a variety of alternate side chains, although there are modest changes in the rate of internal electron transfer from the 6-(S)-cysteinyl FMN to the 4Fe-4S center. The kinetics of electron transfer from the 4Fe-4S center to Fc(+) are sensitive to mutations at position 344. The introduction of smaller side chains (Ala-344, Cys-344, and Gly-344) leads to enhanced rates of electron transfer, and likely reflects shortened electron transfer "pathways" from the 4Fe-4S center to Fc(+). The introduction of larger side chains (Ile-344 and Tyr-344) reduces substantially the rate of electron transfer to Fc(+). Electron transfer to ETF is not affected, to any large extent, by mutation of Val-344. In contrast, mutation of Tyr-442 to Phe, Leu, Cys, and Gly leads to major reductions in the rate of electron transfer to ETF, but not to Fc(+). The data indicate that electron transfer to Fc(+) is via the shortest pathway from the 4Fe-4S center of TMADH to the surface of the enzyme. Val-344 is located at the end of this pathway at the bottom of a small groove on the surface of TMADH, and Fc(+) can penetrate this groove to facilitate good electronic coupling with the 4Fe-4S center. With ETF as an electron acceptor, the observed rate of electron transfer is substantially reduced on mutation of Tyr-442, but not Val-344. We conclude that the flavin of ETF does not penetrate fully the groove on the surface of TMADH, and that electron transfer from the 4Fe-4S center to ETF may involve a longer pathway involving Tyr-442. Mutation of Tyr-442 likely disrupts electron transfer by perturbing the interaction geometry of TMADH and ETF in the productive electron transfer complex, leading to less efficient coupling between the redox centers.
Basran
J
J
Department of Biochemistry, University of Leicester, UK.
Chohan
K K
KK
Sutcliffe
M J
MJ
Scrutton
N S
NS
D002413:Cations, Divalent / Q000737:chemistry; D004579:Electron Transport / Q000235:genetics; D044927:Electron-Transferring Flavoproteins; D005296:Ferrous Compounds / Q000737:chemistry*; D005420:Flavoproteins / Q000737:chemistry*; D007506:Iron-Sulfur Proteins / Q000737:chemistry; D007700:Kinetics; D020630:Methylophilus methylotrophus / Q000201:enzymology / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010089:Oxidoreductases, N-Demethylating / Q000737:chemistry* / Q000235:genetics; D014443:Tyrosine / Q000737:chemistry* / Q000235:genetics; D014633:Valine / Q000737:chemistry* / Q000235:genetics
D002413:Cations, Divalent; D044927:Electron-Transferring Flavoproteins; D005296:Ferrous Compounds; D005420:Flavoproteins; D007506:Iron-Sulfur Proteins; C064804:ferrocenium; D014443:Tyrosine; D010089:Oxidoreductases, N-Demethylating; C023186:trimethylamine dehydrogenase; D014633:Valine