title: Mutation of the heme axial ligand of Escherichia coli succinate-quinone reductase: implications for heme ligation in mitochondrial complex II from yeast.
abstract: A b-type heme is conserved in membrane-bound complex II enzymes (SQR, succinate-ubiquinone reductase). The axial ligands for the low spin heme b in Escherichia coli complex II are SdhC His84 and SdhD His71. E. coli SdhD His71 is separated by 10 residues from SdhD Asp82 and Tyr83 which are essential for ubiquinone catalysis. The same His-10x-AspTyr motif dominates in homologous SdhD proteins, except for Saccharomyces cerevisiae where a tyrosine is at the axial position (Tyr-Cys-9x-AspTyr). Nevertheless, the yeast enzyme was suggested to contain a stoichiometric amount of heme, however, with the Cys ligand in the aforementioned motif acting as heme ligand. In this report, the role of Cys residues for heme coordination in the complex II family of enzymes is addressed. Cys was substituted to the SdhD-71 position and the yeast Tyr71Cys72 motif was also recreated. The Cys71 variant retained heme, although it was high spin, while the Tyr71Cys72 mutant lacked heme. Previously the presence of heme in S. cerevisiae was detected by a spectral peak in fumarate-oxidized, dithionite-reduced mitochondria. Here it is shown that this method must be used with caution. Comparison of bovine and yeast mitochondrial membranes shows that fumarate induced reoxidation of cytochromes in both SQR and the bc1 complex (ubiquinol-cytochrome c reductase). Thus, this report raises a concern about the presence of low spin heme b in S. cerevisiae complex II.
authors.affiliation: Molecular Biology Division, VA Medical Center, Department of Biochemistry and Biophysics, University of California, San Francisco, California 94158, USA.
authors.forename: Elena
authors.initials: E
authors.lastname: Maklashina
authors.forename: Sany
authors.initials: S
authors.lastname: Rajagukguk
authors.forename: William S
authors.initials: WS
authors.lastname: McIntire
authors.forename: Gary
authors.initials: G
authors.lastname: Cecchini
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D017931:DNA Primers / Q000235:genetics; D042963:Electron Transport Complex II / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006418:Heme / Q000737:chemistry; D066298:In Vitro Techniques; D007700:Kinetics; D008024:Ligands; D008928:Mitochondria / Q000201:enzymology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013053:Spectrophotometry
chemical_list: D017931:DNA Primers; D029968:Escherichia coli Proteins; D008024:Ligands; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D006418:Heme; D042963:Electron Transport Complex II