Isolation and characterization of a new class of cytochrome d terminal oxidase mutants of Escherichia coli

Oden, Kristine L.; Gennis, Robert B.

doi:10.1128/jb.173.19.6174-6183.1991

Cited by 17 publications

(11 citation statements)

References 48 publications

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“…GK100 and GK101 were constructed in this work as described below. Strain GO 105 is a partial deletion of the cyd operon with a corresponding insertion of a kanamycin-resistance cartridge (Calhoun et al, 1993b;Oden & Gennis, 1991). This strain contains an undefined mutation in the cyo operon preventing production of cytochrome her.…”

Section: Methodsmentioning

confidence: 99%

“…GO105 was, therefore, constructed and used for the complementation analysis. This strain contains the partially deleted chromosomal cyd operon of GO 102 (Calhoun et al, 1993b;Oden & Gennis, 1991) 'but is also recA, thus preventing homologous recombination. This strain was constructed by transducing reck into GO 102 (Calhoun et al, 1993b;Oden & Gennis, 1991).…”

Section: Methodsmentioning

confidence: 99%

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Methionine-393 is an axial ligand of the heme b558 component of the cytochrome bd ubiquinol oxidase from Escherichia coli

Kaysser¹,

Ghaim²,

Georgiou³

et al. 1995

Biochemistry

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The cytochrome bd oxidase is one of two terminal oxidases in the aerobic respiratory chain of Escherichia coli. The complex is composed of two subunits (I and II) and three heme prosthetic groups (heme b558, heme b595, and a chlorin, called heme d). Both subunits are located within the bacterial cytoplasmic membrane, and each has multiple putative transmembrane helices. Heme b558 is a six-coordinate, low-spin heme component of the oxidase which has been shown to be contained within subunit I and has been implicated in the oxidation of the substrate, ubiquinol-8, in the cytoplasmic membrane. Previous site-directed mutagenesis studies identified His186, predicted to be near the periplasmic side of transmembrane helix D of subunit I, as one of the axial ligands of heme b558. Since mutagenesis of none of the other histidines in subunit I perturbs heme b558, it was concluded that this heme cannot have bis(histidine) ligation. In this work, the properties of 14 mutants are reported, including substitutions for each of 10 methionine residues within subunit I. Among this set of mutants, only the replacement of M393 perturbs heme b558. Replacement of M393 by leucine results in the conversion of heme b558 to a high-spin state. Surprisingly, the M393L mutation does not eliminate enzymatic activity, and the mutant oxidase has sufficient turnover to support aerobic growth of the cells. The addition of imidazole to the purified M393L oxidase converts heme b558 back to a low-spin configuration. The data strongly suggest that the sixth axial ligand of heme b558 is methionine-393, and that this heme, therefore, has histidine-methionine ligation. The results are consistent with recent cryogenic near-infrared magnetic circular dichroism spectra that also indicate histidine-methionine ligation of heme b558.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Methionine-393 is an axial ligand of the heme b558 component of the cytochrome bd ubiquinol oxidase from Escherichia coli

Kaysser¹,

Ghaim²,

Georgiou³

et al. 1995

Biochemistry

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“…Within the subfamily of bd -type oxygen reductases which have the “long Q-loop”, Arg100, Asp29, and Asp120 of subunit II are totally conserved and Asp58 (subunit II of E. coli cytochrome bd -I) is either an aspartate or glutamate [213]. The N-terminal portion of subunit II has been suggested to be involved in the binding of heme d /heme b 595 [213,254]. …”

Section: Proposed Structurementioning

confidence: 99%

The cytochrome bd respiratory oxygen reductases

Borisov

Gennis

Hemp

et al. 2011

Biochimica et Biophysica Acta (BBA) - Bioenergetics

453

533

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Summary Cytochrome bd is a respiratory quinol:O2 oxidoreductase found in many prokaryotes, including a number of pathogens. The main bioenergetic function of the enzyme is the production of a proton motive force by the vectorial charge transfer of protons. The sequences of cytochromes bd are not homologous to those of the other respiratory oxygen reductases, i.e., the heme-copper oxygen reductases or alternative oxidases (AOX). Generally, cytochromes bd are noteworthy for their high affinity for O2 and resistance to inhibition by cyanide. In E. coli, for example, cytochrome bd (specifically, cytochrome bd-I) is expressed under O2-limited conditions. Among the members of the bd-family are the so-called cyanide-insensitive quinol oxidases (CIO) which often have a low content of the eponymous heme d but, instead, have heme b in place of heme d in at least a majority of the enzyme population. However, at this point, no sequence motif has been identified to distinguish cytochrome bd (with a stoichiometric complement of heme d) from an enzyme designated as CIO. Members of the bd-family can be subdivided into those which contain either a long or a short hydrophilic connection between transmembrane helices 6 and 7 in subunit I, designated as the Q-loop. However, it is not clear whether there is a functional consequence of this difference. This review summarizes current knowledge on the physiological functions, genetics, structural and catalytic properties of cytochromes bd. Included in this review are descriptions of the intermediates of the catalytic cycle, the proposed site for the reduction of O2, evidence for a proton channel connecting this active site to the bacterial cytoplasm, and the molecular mechanism by which a membrane potential is generated.

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“…These results suggest that insertion of the low-spin heme B and of the heme BD binuclear center occur independently from each other, but formation of the binuclear center requires the presence of both subunits. It has been suggested that SUI may interact with SUII to form an interface in which the heme BD cofactors bind (246).…”

Section: Maturation Of Terminal Oxidasesmentioning

confidence: 99%