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

Journal Article · · Journal of Biological Chemistry
 [1];  [2];  [1];  [1];  [2]; ORCiD logo [1]
  1. Vanderbilt Univ., Nashville, TN (United States)
  2. Univ. of California, San Francisco, CA (United States); Veterans Affairs Medical Center, San Francisco, CA (United States)
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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.

Research Organization:
Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Organization:
USDOE Office of Science (SC)
Grant/Contract Number:
AC02-06CH11357; R01GM105404; S10OD018483; 085P1000817
OSTI ID:
1375349
Journal Information:
Journal of Biological Chemistry, Vol. 292, Issue 31; ISSN 0021-9258
Publisher:
American Society for Biochemistry and Molecular BiologyCopyright Statement
Country of Publication:
United States
Language:
ENGLISH
Citation Metrics:
Cited by: 11 works
Citation information provided by
Web of Science

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Cited By (5)

Crystal structure of bacterial succinate:quinone oxidoreductase flavoprotein SdhA in complex with its assembly factor SdhE journal March 2018
The unassembled flavoprotein subunits of human and bacterial complex II have impaired catalytic activity and generate only minor amounts of ROS journal April 2018
The microenvironment surrounding FAD mediates its conversion to 8-formyl-FAD in Aspergillus oryzae RIB40 formate oxidase journal February 2019
Crystal structure of an assembly intermediate of respiratory Complex II journal April 2018
Crystal structure of an assembly intermediate of respiratory Complex II journal January 2018

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