Role of His residues in Bacillus subtilis cytochrome b558 for haem binding and assembly of succinate: quinone oxidoreductase (complex II).
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.
Fridén
H
H
Department of Microbiology, University of Lund, Sweden.
Hederstedt
L
L
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003062:Codon; D003573:Cytochrome b Group / Q000378:metabolism*; D004247:DNA / Q000235:genetics; D042963:Electron Transport Complex II; D006418:Heme / Q000378:metabolism*; D006639:Histidine / Q000235:genetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*; D009154:Mutation; D019255:NADPH Oxidases; D010088:Oxidoreductases / Q000378:metabolism*; D011487:Protein Conformation; D013385:Succinate Dehydrogenase / Q000378:metabolism*
D003062:Codon; D003573:Cytochrome b Group; D009097:Multienzyme Complexes; D006418:Heme; D006639:Histidine; D004247:DNA; C036888:cytochrome b558; D010088:Oxidoreductases; D042963:Electron Transport Complex II; D013385:Succinate Dehydrogenase; D019255:NADPH Oxidases