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PDBsum entry 3cls
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Electron transport
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PDB id
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3cls
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References listed in PDB file
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Key reference
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Title
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Probing the dynamic interface between trimethylamine dehydrogenase (tmadh) and electron transferring flavoprotein (etf) in the tmadh-2etf complex: role of the arg-Alpha237 (etf) and tyr-442 (tmadh) residue pair.
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Authors
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S.G.Burgess,
H.L.Messiha,
G.Katona,
S.E.Rigby,
D.Leys,
N.S.Scrutton.
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Ref.
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Biochemistry, 2008,
47,
5168-5181.
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PubMed id
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Abstract
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We have used multiple solution state techniques and crystallographic analysis to
investigate the importance of a putative transient interaction formed between
Arg-alpha237 in electron transferring flavoprotein (ETF) and Tyr-442 in
trimethylamine dehydrogenase (TMADH) in complex assembly, electron transfer, and
structural imprinting of ETF by TMADH. We have isolated four mutant forms of ETF
altered in the identity of the residue at position 237 (alphaR237A, alphaR237K,
alphaR237C, and alphaR237E) and with each form studied electron transfer from
TMADH to ETF, investigated the reduction potentials of the bound ETF cofactor,
and analyzed complex formation. We show that mutation of Arg-alpha237
substantially destabilizes the semiquinone couple of the bound FAD and impedes
electron transfer from TMADH to ETF. Crystallographic structures of the mutant
ETF proteins indicate that mutation does not perturb the overall structure of
ETF, but leads to disruption of an electrostatic network at an ETF domain
boundary that likely affects the dynamic properties of ETF in the crystal and in
solution. We show that Arg-alpha237 is required for TMADH to structurally
imprint the as-purified semiquinone form of wild-type ETF and that the ability
of TMADH to facilitate this structural reorganization is lost following (i)
redox cycling of ETF, or simple conversion to the oxidized form, and (ii)
mutagenesis of Arg-alpha237. We discuss this result in light of recent apparent
conflict in the literature relating to the structural imprinting of wild-type
ETF. Our studies support a mechanism of electron transfer by conformational
sampling as advanced from our previous analysis of the crystal structure of the
TMADH-2ETF complex [Leys, D. , Basran, J. , Sutcliffe, M. J., and Scrutton, N.
S. (2003) Nature Struct. Biol. 10, 219-225] and point to a key role for the
Tyr-442 (TMADH) and Arg-alpha237 (ETF) residue pair in transiently stabilizing
productive electron transfer configurations. Our work also points to the
importance of Arg-alpha237 in controlling the thermodynamics of electron
transfer, the dynamics of ETF, and the protection of reducing equivalents
following disassembly of the TMADH-2ETF complex.
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