Transmembrane topology and axial ligands to hemes in the cytochrome b subunit of Bacillus subtilis succinate:menaquinone reductase.
The membrane-anchoring subunit of Bacillus subtilis succinate:menaquinone reductase is a protein of 202 residues containing two protoheme IX groups with bis-histidine axial ligation. Residues His13, His28, His70, His113, and His155 are the possible heme ligands. The transmembrane topology of this cytochrome was analyzed using fusions to alkaline phosphatase. The results support a proposed model with five transmembrane polypeptide segments and the N-terminus exposed to the cytoplasm. Mutant B. subtilis cytochromes containing a His13-->Tyr, a His28-->Tyr, and a His113-->Tyr mutation, respectively, were produced in Escherichia coli, partially purified, and analyzed. In addition, succinate: menaquinone reductase containing the His13-->Tyr mutation in the anchor subunit was overproduced in B. subtilis, purified, and characterized. The data demonstrate that His13 is not an axial heme ligand. Thermodynamic and spectroscopic properties of the cytochrome are, however, affected by the His13-->Tyr mutation; compared to wild type, the redox potentials of both hemes are negatively shifted and the gmax signal in the EPR spectrum of the high-potential heme is shifted from 3.68 to 3.50. From the combined results we conclude that His28 and His113 function as axial ligands to the low-potential heme, which is located in the membrane near the outer surface of the cytoplasmic membrane. Residues His70 and His155 ligate the high-potential heme, which is positioned close to His13 in the protein, near the inner surface of the membrane.
Hägerhäll
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C
Department of Microbiology, Lund University, Sweden.
Fridén
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H
Aasa
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Hederstedt
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D000469:Alkaline Phosphatase / Q000737:chemistry / Q000235:genetics; D001412:Bacillus subtilis / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D003573:Cytochrome b Group / Q000737:chemistry* / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D042963:Electron Transport Complex II; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006418:Heme / Q000737:chemistry; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D019255:NADPH Oxidases; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics; D017354:Point Mutation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D013385:Succinate Dehydrogenase / Q000737:chemistry* / Q000235:genetics
D003573:Cytochrome b Group; D017931:DNA Primers; D004269:DNA, Bacterial; D008024:Ligands; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; D006418:Heme; C036888:cytochrome b558; D010088:Oxidoreductases; D042963:Electron Transport Complex II; D013385:Succinate Dehydrogenase; D019255:NADPH Oxidases; D000469:Alkaline Phosphatase