Differential Coupling through Val-344 and Tyr-442 of Trimethylamine Dehydrogenase in Electron Transfer Reactions with Ferricenium Ions and Electron Transferring Flavoprotein

Abstract

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.

Electron Transfer and Conformational Change in Complexes of Trimethylamine Dehydrogenase and Electron Transferring Flavoprotein

“…Our difference spectroscopy studies reported in this paper provide direct evidence for impaired rates of complex assembly with the mutant TMADH enzymes (Table I), thereby supporting our previous kinetic model ( (18), see Discussion).…”

“…3 in Ref. 18. Incubation of ETF sq and ETF ox with TMADH ox leads to a gradual ϳ4-fold increase in ETF flavin fluorescence, with a half-life of about 30 min and reaching a plateau after about 2 h (Fig.…”

“…The spectral changes accompanying assembly of both the Y442G TMADH⅐ETF ox and TMADH⅐ETF sq complexes are observable in stopped-flow studies (Table I). Furthermore, formation of the Y442G TMADH⅐ETF ox and TMADH⅐ETF sq complexes occurs on much longer time scales than the corresponding rates of electron transfer when normalized to the equivalent concentra-tions of the component proteins (Table I ( 18)), suggesting that electron transfer occurs in a thermodynamically metastable electron transfer complex. Our work described here thus establishes that the rates of complex formation with Y442F, Y442L, or Y442G TMADH and ETF sq are substantially compromised, which probably accounts for the reduction in electron transfer rate in these mutant complexes.…”

“…Mutation of Tyr-442 on the surface of TMADH compromises substantially the rate of electron transfer from the 4Fe-4S center of TMADH to the FAD of ETF ((18), Table I). We conjectured previously that the compromised rates of electron transfer observed with the mutant TMADH enzymes are attributed to impaired rates of conformational change/structural realignment in the complex required for subsequent electron transfer (18) and that the electron transfer event could be described by the following kinetic scheme (Reaction 2):…”

“…Following substrate reduction of TMADH, electrons are transferred sequentially from the FMN cofactor to the 4Fe-4S center and, subsequently, in an interprotein electron transfer reaction to the FAD of electron transferring flavoprotein (ETF) (17,18). ETF from M. methylotrophus is a heterodimer with subunit masses of 34 and 29 kDa (19).…”