Two Hydrophobic Subunits Are Essential for the Heme b Ligation and Functional Assembly of Complex II (Succinate-Ubiquinone Oxidoreductase) from Escherichia coli

Abstract

Complex II (succinate-ubiquinone oxidoreductase) from Escherichia coli is composed of four nonidentical subunits encoded by the sdhCDAB operon. Gene products of sdhC and sdhD are small hydrophobic subunits that anchor the hydrophilic catalytic subunits (flavoprotein and iron-sulfur protein) to the cytoplasmic membrane and are believed to be the components of cytochrome b 556 in E. coli complex II. In the present study, to elucidate the role of two hydrophobic subunits in the heme b ligation and functional assembly of complex II, plasmids carrying portions of the sdh gene were constructed and introduced into E. coli MK3, which lacks succinate dehydrogenase and fumarate reductase activities. The expression of polypeptides with molecular masses of about 19 and 17 kDa was observed when sdhC and sdhD were introduced into MK3, respectively, indicating that sdhC encodes the large subunit (cybL) and sdhD the small subunit (cybS) of cytochrome b 556 . An increase in cytochrome b content was found in the membrane when sdhD was introduced, while the cytochrome b content did not change when sdhC was introduced. However, the cytochrome b expressed by the plasmid carrying sdhD differed from cytochrome b 556 in its CO reactivity and red shift of the ␣ absorption peak to 557.5 nm at 77 K. Neither hydrophobic subunit was able to bind the catalytic portion to the membrane, and only succinate dehydrogenase activity, not succinateubiquinone oxidoreductase activity, was found in the cytoplasmic fractions of the cells. In contrast, significantly higher amounts of cytochrome b 556 were expressed in the membrane when sdhC and sdhD genes were both present, and the catalytic portion was found to be localized in the membrane with succinate-ubiquinone oxidoreductase and succinate oxidase activities. These results strongly suggest that both hydrophobic subunits are required for heme insertion into cytochrome b 556 and are essential for the functional assembly of E. coli complex II in the membrane. Accumulation of the catalytic portion in the cytoplasm was found when sdhCDAB was introduced into a heme synthesis mutant, suggesting the importance of heme in the assembly of E. coli complex II.Complex II (succinate-ubiquinone oxidoreductase) is a tricarboxylic acid cycle enzyme associated with the inner membrane of mitochondria or the cytoplasmic membrane of bacteria (1, 2). Complex II in Escherichia coli is encoded by the sdhCDAB operon (3, 4) and contains four nonidentical subunits and five prosthetic groups (5, 6). The largest hydrophilic subunit (flavoprotein; Fp), 1 encoded by sdhA, has covalently bound FAD, and the second largest subunit (iron-sulfur protein; Ip), encoded by sdhB, contains three different types of iron-sulfur clusters termed S-1[2Fe-2S], S-2[4Fe-4S], and S-3[3Fe-4S]. Two hydrophobic heme b-containing subunits are encoded by sdhC and sdhD, and at least the sdhC product is a component of cytochrome b 556 (7).Generally, the Fp and Ip subunits comprise the catalytic portion of complex II and catalyze electron transfer from suc...

Identification of Quinone-binding and Heme-ligating Residues of the Smallest Membrane-anchoring Subunit (QPs3) of Bovine Heart Mitochondrial Succinate:Ubiquinone Reductase

“…When hemin chloride was added to a 1:1 mixture of recombinant QPs1 and QPs3, the amount of cytochrome b 560 restoration equals the sum of cytochrome b 560 restored by the individual recombinant proteins, suggesting that each of these proteins can provide bishistidine ligands for cytochrome b 560 in bovine heart mitochondrial succinate:Q reductase. This differs from the report that cytochrome b 556 in E. coli succinate:Q reductase is ligated to two histidine residues located, respectively, at SdhC and SdhD (14).…”

“…The ligand for this cytochrome has been identified as bishistidine (12). However, unknown is which QPs subunit is involved in heme b 560 ligation, and whether the bishistidine ligands are provided by a single subunit or by two different subunits, as reported for cytochrome b 556 of E. coli succinate:Q reductase (14,15). Because recombinant QPs3 contains little cytochrome b 560 heme, the involvement of QPs3 in heme ligation was investigated by testing the ability of recombinant QPs3 to reconstitute in vitro with hemin chloride to form cytochrome b 560 .…”

“…Both the membraneanchoring subunits (SdhC and SdhD) in the E. coli enzyme are involved in heme ligation of cytochrome b 556 (14) . His-84 of the SdhC and His-71 of the SdhD were identified as ligands for cytochrome b 556 (15).…”

Heme Axial Ligation by the Highly Conserved His Residues in Helix II of Cytochrome b (NarI) of Escherichia coliNitrate Reductase A (NarGHI)

“…Therefore, heme insertion must depend on some aspects of the quaternary interaction within NarGHI. Such a phenomenon has already been described for the assembly of the Bacillus subtilis and E. coli succinate:quinone oxidoreductases (34,35) and for the cytochrome d terminal oxidase complex of E. coli (36). In the latter case, heme insertion in the different membrane-bound subunits can only occur after the complete formation of the cytochrome bd oxidase complex.…”

Mitochondrial respiratory chain complexes: apoptosis sensors mutated in cancer?

“…As this subcomplex is still enzymatically active (Nakamura et al, 1996;Cecchini, 2003), it can remove electrons from its substrate succinate and transfer them to molecular oxygen. This uncontrolled enzymatic activity thereby generates excessive ROS for apoptosis induction (Albayrak et al, 2003;Lemarie et al, 2011).…”