The Interaction of Trimethylamine Dehydrogenase and Electron-transferring Flavoprotein

Weiwei Shi,John Mersfelder,Russ Hille

doi:10.1074/jbc.m500582200

Weiwei Shi, John Mersfelder + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m500582200

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Abstract

The interaction between the physiological electron transfer partners trimethylamine dehydrogenase (TMADH) and electron-transferring flavoprotein (ETF) from Methylophilus methylotrophus has been examined with particular regard to the proposal that the former protein "imprints" a conformational change on the latter. The results indicate that the absorbance change previously attributed to changes in the environment of the FAD of ETF upon binding to TMADH is instead caused by electron transfer from partially reduced, as-isolated TMADH to ETF. Prior treatment of the as-isolated enzyme with the oxidant ferricenium essentially abolishes the observed spectral change. Further, when the semiquinone form of ETF is used instead of the oxidized form, the mirror image of the spectral change seen with as-isolated TMADH and oxidized ETF is observed. This is attributable to a small amount of electron transfer in the reverse of the physiological direction. Kinetic determination of the dissociation constant and limiting rate constant for electron transfer within the complex of (reduced) TMADH with (oxidized) ETF is reconfirmed and discussed in the context of a recently proposed model for the interaction between the two proteins that involves "structural imprinting" of ETF.

Highlights

Trimethylamine dehydrogenase (TMADH,1 EC 1.5.99.7) isolated from the bacterium Methylophilus methylotrophus is an iron-sulfur-containing flavoprotein consisting of two identical, catalytically independent subunits
The Binding of As-isolated trimethylamine dehydrogenase (TMADH) to Oxidized electron-transferring flavoprotein (ETF) and ETF Semiquinone—The spectral change observed upon mixing TMADHai and ETFox is shown in Fig. 1A and is in good agreement with that reported previously [10]
The essentially mirror image relationship of the spectral changes seen on mixing TMADHai with ETFox on the one hand, and TMADHai with ETF semiquinone (ETFsq) on the other, suggests strongly that these changes do not arise from a change in the environment of the FAD of ETF

Summary

Electron Transfer between TMADH and ETF

A donor-acceptor distance of ϳ12 Å; approximately the extent to which the [4Fe-4S] is buried in TMADH. Whereas results with the mutant TMADH forms were largely consistent with the earlier study, the wild-type enzyme was found to exhibit kinetics with a linear dependence on [ETF] rather than the hyperbolic dependence found in the two earlier studies. Based on this observation, a model was proposed in which mutations at Tyr442 had very large effects on the rate of electron transfer from the iron-sulfur center of TMADH to the FAD of ETF, the effect was masked by a rate-limiting conformational change with the wild-type enzyme.

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION