Localization of histidine residues responsible for heme axial ligation in cytochrome b556 of complex II (succinate:ubiquinone oxidoreductase) in Escherichia coli.
Complex II (succinate:ubiquinone oxidoreductase) from Escherichia coli contains four different subunits. Two of the subunits (SDHC and SDHD) are hydrophobic and anchor the two more hydrophilic (flavin and iron-sulfur) subunits (SDHA and SDHB) to the cytoplasmic membrane. Previous studies have shown that the complex of SDHC/SDHD is required to maintain the heme B component of the enzyme and that the heme B is ligated to the protein by two histidine ligands. In the current work, the histidines within SDHC and SDHD have been systematically mutated. SDHC-His91 and SDHD-His14 were eliminated as potential ligands by these studies. SDHC-His84 and SDHD-His71 have been identified as the most likely heme axial ligands in the E. coli enzyme, suggesting that the heme bridges these two subunits in the membrane. Furthermore, the results show that the four-subunit Complex II assembles and retains function despite the absence of the heme B prosthetic group in the membrane. The results do not rule out completely SDHC-His30 as a candidate for heme ligation, but do show that mutation at this position prevents assembly of Complex II in the membrane.
Vibat
C R
CR
Department of Biochemistry, University of Illinois at Urbana-Champaign 61801, USA.
Cecchini
G
G
Nakamura
K
K
Kita
K
K
Gennis
R B
RB
D000332:Aerobiosis; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics*; D002847:Chromatography, Agarose; D002851:Chromatography, High Pressure Liquid; D003573:Cytochrome b Group / Q000235:genetics / Q000378:metabolism*; D042963:Electron Transport Complex II; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D006418:Heme / Q000235:genetics / Q000378:metabolism*; D006639:Histidine / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000096:biosynthesis / Q000378:metabolism; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013385:Succinate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D003573:Cytochrome b Group; D008565:Membrane Proteins; D009097:Multienzyme Complexes; C018256:cytochrome b556; D006418:Heme; D006639:Histidine; D010088:Oxidoreductases; D042963:Electron Transport Complex II; D013385:Succinate Dehydrogenase