Role of His residues in Bacillus subtilis cytochrome b558 for haem binding and assembly of succinate: quinone oxidoreductase (complex II)

Abstract

Cytochrome b558 in the cytoplasmic membrane of Bacillus subtilis constitutes the anchor and electron acceptor to the flavoprotein (Fp) and iron-sulphur protein (Ip) in succinate:quinone oxidoreductase, and seemingly contains two haem groups. EPR and MCD spectroscopic data indicate bis-imidazole ligation of the haem. Apo-cytochrome was found in the membrane fraction of haem-deficient B. subtilis, suggesting that during biogenesis of the oxidoreductase the cytochrome b558 polypeptide is embedded into the membrane prior to the incorporation of haem and subsequent binding of Fp and Ip. The six His residues in cytochrome b558 were individually changed to Tyr to attempt identification of residues serving as haem axial ligands and to analyse the role of His residues for assembly and function of the oxidoreductase. From the properties of the mutants, His-47 can be excluded as a haem ligand. The remaining His residues (at positions 13, 28, 70, 113 and 155) are located in or close to four predicted transmembrane segments. The Tyr-28 and Tyr-70 mutant proteins appeared to lack one of the two haems. Only the Tyr-13 and Tyr-47 mutant cytochromes were found to function as anchors for Fp and Ip, but the Tyr-13 mutant cytochrome assembles into an enzymatically defective succinate:quinone oxidoreductase. It is concluded from a combination of the experimental findings, sequence comparisons and membrane topology data that His-28, His-70 and His-155 are probably haem axial ligands in a dihaem cytochrome b558. His-70 and His-155 may be ligands to the same haem.

A structural moDAl for the membrane‐integral domain of succinate:quinone oxidoreductases

“…The ligand histidine residues have been identified in the primary sequence of the B. subtilis and E. coli SQR anchor polypeptides by site directed mutagenesis analysis [12,16,17]. These residues are located in predicted transmembrane segments as shown in Fig.…”

Spectroscopic identification of the axial ligands of cytochrome b560 in bovine heart succinate‐ubiquinone reductase

“…Discussion 1600 nm [14,16]. Moreover, mutagenesis studies of specific histidine residues in B. subtilis cytochrome b558 have identified four The primary objectives of this work was twofold: to establish histidines in hydrophobic segments that are essential for heme the axial ligation of cytochrome b560 in SQR and to investigate ligation and correct assembly of SQR [30]. Hence the available the origin of the changes in properties of this prosthetic group mutagenesis data provides additional support for a His/His that accompany isolation of the QPs.…”

Identification of the axial heme ligands of cytochrome b556 in succinate: Ubiquinone oxidoreductase from Escherichia coli

“…A combination of EPR and near-infrared MCD spectroscopies [12] have shown that each of the heroes associated with the hydrophobic subunit (cytochrome b55s) of this enzyme has bis(histidine) ligation [11]. Subsequent site-directed mutagenesis studies have resulted in a postulated model in which specific histidines within this subunit have been assigned to the cytochrome components [10]. The data in this paper show that the single protoheme component of the E. coli succinate dehydrogenase also has bis(histidine) ligation.…”

“…the heme ligands within this family of enzymes have been those with the succinate dehydrogenase from B. subtilis [10,11]. A combination of EPR and near-infrared MCD spectroscopies [12] have shown that each of the heroes associated with the hydrophobic subunit (cytochrome b55s) of this enzyme has bis(histidine) ligation [11].…”