PMID:1309808
| Citation |
Minagawa, J, Mogi, T, Gennis, RB and Anraku, Y (1992) Identification of heme and copper ligands in subunit I of the cytochrome bo complex in Escherichia coli. J. Biol. Chem. 267:2096-104 |
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| Abstract |
The cytochrome bo complex is a terminal ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli (Kita, K., Konishi, K., and Anraku, Y. (1984) J. Biol. Chem. 259, 3368-3374) and functions as a proton pump. It belongs to the heme-copper oxidase superfamily with the aa3-type cytochrome c oxidases in mitochondria and aerobic bacteria. In order to identify ligands of hemes and copper, we have substituted eight conserved histidines in subunit I by alanine and, in addition, His-106, -284, and -421 by glutamine and methionine. Western immunoblotting analysis showed that all the mutations do not affect the expression level of subunit I in the cytoplasmic membrane, indicating that these histidines are not crucial for its stability. A single copy expression vector carrying a single mutation at the invariant histidines, His-106, His-284, His-333, His-334, His-419, and His-421, of subunit I was unable to support the aerobic growth of a strain in which the chromosomal terminal oxidase genes (the cyo and cyd operons) have been deleted. The same mutations caused a complete loss of ubiquinol oxidase activity of the partially purified enzymes. Spectroscopic analysis of mutant oxidases in the cytoplasmic membrane revealed that substitutions of His-106 and -421 specifically eliminated a 563.5 nm peak of the low spin heme and that replacements of His-106, -284, and -419 reduced the extent of the CO-binding high spin heme. These spectroscopic properties of mutant oxidases were further confirmed with partially purified preparations. Atomic absorption analysis showed that substitutions of His-106, -333, -334, and -419 eliminated CuB almost completely. Based on these findings, we conclude that His-106 and -421 function as the axial ligands of the low spin heme and His-284 is a possible ligand of the high spin heme. His-333, -334, and -419 residues are attributed to the ligands of CuB. We present a helical wheel model of the redox center in subunit I, which consists of the membrane-spanning regions II, VI, VII, and X, and discuss the implications of the model. |
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| Keywords |
Aerobiosis; Amino Acid Sequence; Animals; Base Sequence; Binding Sites; Copper/metabolism; DNA, Bacterial/genetics; Dithionite/pharmacology; Electron Transport Complex IV/chemistry; Electron Transport Complex IV/genetics; Electron Transport Complex IV/metabolism; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli/growth & development; Genetic Complementation Test; Heme/metabolism; Humans; Ligands; Macromolecular Substances; Models, Structural; Molecular Sequence Data; Mutagenesis, Site-Directed; Oligodeoxyribonucleotides; Oxidation-Reduction; Protein Conformation; Recombinant Proteins/chemistry; Recombinant Proteins/metabolism; Restriction Mapping; Sequence Homology, Nucleic Acid |
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