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J Biol Chem. 2017 Aug 4;292(31):12921-12933. doi: 10.1074/jbc.M117.795120. Epub 2017 Jun 14.

Structural and biochemical analyses reveal insights into covalent flavinylation of the Escherichia coli Complex II homolog quinol:fumarate reductase.

Author information

1
From the Graduate Program in Chemical and Physical Biology.
2
the Molecular Biology Division, Veterans Affairs Medical Center, San Francisco, California 94121, and.
3
the Department of Biochemistry and Biophysics, University of California, San Francisco, California 94158.
4
Departments of Pharmacology.
5
Cell and Developmental Biology, and.
6
the Molecular Biology Division, Veterans Affairs Medical Center, San Francisco, California 94121, and Gary.Cecchini@ucsf.edu.
7
Departments of Pharmacology, tina.iverson@vanderbilt.edu.
8
Biochemistry.
9
Center for Structural Biology, and.
10
Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, Tennessee 37232.

Abstract

The Escherichia coli Complex II homolog quinol:fumarate reductase (QFR, FrdABCD) catalyzes the interconversion of fumarate and succinate at a covalently attached FAD within the FrdA subunit. The SdhE assembly factor enhances covalent flavinylation of Complex II homologs, but the mechanisms underlying the covalent attachment of FAD remain to be fully elucidated. Here, we explored the mechanisms of covalent flavinylation of the E. coli QFR FrdA subunit. Using a ΔsdhE E. coli strain, we show that the requirement for the assembly factor depends on the cellular redox environment. We next identified residues important for the covalent attachment and selected the FrdAE245 residue, which contributes to proton shuttling during fumarate reduction, for detailed biophysical and structural characterization. We found that QFR complexes containing FrdAE245Q have a structure similar to that of the WT flavoprotein, but lack detectable substrate binding and turnover. In the context of the isolated FrdA subunit, the anticipated assembly intermediate during covalent flavinylation, FrdAE245 variants had stability similar to that of WT FrdA, contained noncovalent FAD, and displayed a reduced capacity to interact with SdhE. However, small-angle X-ray scattering (SAXS) analysis of WT FrdA cross-linked to SdhE suggested that the FrdAE245 residue is unlikely to contribute directly to the FrdA-SdhE protein-protein interface. We also found that no auxiliary factor is absolutely required for flavinylation, indicating that the covalent flavinylation is autocatalytic. We propose that multiple factors, including the SdhE assembly factor and bound dicarboxylates, stimulate covalent flavinylation by preorganizing the active site to stabilize the quinone-methide intermediate.

KEYWORDS:

Complex II; bioenergetics; flavin adenine dinucleotide (FAD); membrane enzyme; oxidation-reduction (redox); protein assembly; protein structure; respiratory chain

PMID:
28615448
PMCID:
PMC5546032
DOI:
10.1074/jbc.M117.795120
[Indexed for MEDLINE]
Free PMC Article

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