Identification of the functional domains in heme O synthase. Site-directed mutagenesis studies on the cyoE gene of the cytochrome bo operon in Escherichia coli.
The cytochrome bo complex is a terminal ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli and is encoded by the cyoABCDE operon. Recently, we have demonstrated that heme O at the high-spin heme-binding site is essential for redox-coupled proton pumping by the oxidase and suggested that the cyoE gene encodes a novel enzyme for heme O biosynthesis, protoheme IX farnesyltransferase (heme O synthase) (Saiki, K., Mogi, T., and Anraku, Y. (1992) Biochem. Biophys. Res. Commun. 189, 1491-1497). This study was focused to define the catalytic domain(s) of the CyoE protein via a site-directed mutagenesis approach. We have individually substituted 40 amino acid residues including 22 invariant residues with alanines and found that 23 mutant oxidases were nonfunctional and exhibited a specific loss of the CO binding activity at the site of the high-spin heme. Characterizations of the purified D65A, Y120A, and W172A mutant oxidases, which represent the mutations of different topological domains, revealed that their defects are attributable to substitution of protoheme IX for heme O present in the high-spin heme-binding site. Based on the above observations, we suggest that the conserved amino acid residues present in the cytoplasmic loops II/III and IV/V are part of the catalytic center of heme O synthase.
Saiki
K
K
Department of Plant Sciences, Graduate School of Science, University of Tokyo, Japan.
Mogi
T
T
Hori
H
H
Tsubaki
M
M
Anraku
Y
Y
D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D003300:Copper / Q000378:metabolism; D003573:Cytochrome b Group; D003580:Cytochromes / Q000235:genetics*; D003593:Cytoplasm / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D006418:Heme / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009876:Operon; D013057:Spectrum Analysis; D014166:Transferases / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D003573:Cytochrome b Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; D006418:Heme; D003300:Copper; C017168:cytochrome bo, E coli; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C078356:CyoE protein, Bacteria; C489727:CyoE protein, E coli