In-vitro, SDH5-dependent flavinylation of immobilized human respiratory complex II flavoprotein.
Mitochondrial Complex II (Succinate: ubiquinone oxidoreductase) has a covalently bound FAD cofactor in its largest subunit (SDHA), which accepts electrons from oxidation of succinate during catalysis. The mechanism of flavin attachment, and factors involved, have not been fully elucidated. The recent report of an assembly factor SDH5 (SDHAF2, SDHE) required for flavinylation (Hao et al., 2009 Science 325, 1139-1142) raises the prospect of achieving flavinylation in a completely defined system, which would facilitate elucidation of the precise role played by SDH5 and other factors. At this time that goal has not been achieved, and the actual function of SDH5 is still unknown. We have developed a procedure for in-vitro flavinylation of recombinant human apo-SDHA, immobilized on Ni-IMAC resin by a His tag, in a chemically defined medium. In this system flavinylation has a pH optimum of 6.5 and is completely dependent on added SDH5. The results suggest that FAD interacts noncovalently with SDHA in the absence of SDH5. This system will be useful in understanding the process of flavinylation of SDHA and the role of SDH5 in this process.
Dept. of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA.
Lala
L
Zafreen
Dept. of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA.
Nancy
N
Walker-Kopp
Dept. of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA.
Li-Shar
LS
Huang
Dept. of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA. Electronic address: berrye@upstate.edu.
Edward
E
Berry
D002384:Catalysis; D004305:Dose-Response Relationship, Drug; D042963:Electron Transport Complex II / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005415:Flavins / Q000737:chemistry*; D005420:Flavoproteins / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D055767:Immobilized Proteins / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000378:metabolism*; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D021122:Protein Subunits / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013696:Temperature
D005415:Flavins; D005420:Flavoproteins; D055767:Immobilized Proteins; D024101:Mitochondrial Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; C543341:SDHAF2 protein, human; C478189:respiratory complex II; D005182:Flavin-Adenine Dinucleotide; D006639:Histidine; D042963:Electron Transport Complex II; C511268:SDHA protein, human
Assembly factor; Electron transfer complex; Enzyme assembly; Flavin adenine dinucleotide; Post-translational modification; Succinate dehydrogenase