Expression Studies on the ba3 Quinol Oxidase from Paracoccus Denitrificans A bb3 Variant is Enzymatically Inactive

Zickermann, Irmela; Tautu, Oltea S.; Link, Thomas; Korn, Marcus; Ludwig, Bernd; Richter, Oliver‐Matthias H.

doi:10.1111/j.1432-1033.1997.00618.x

Cited by 19 publications

(32 citation statements)

References 36 publications

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“…In this study we address the question of heme composition of the E. coli oxidase when expressed in a P. denitrificans host, and show that the purified enzyme exhibits a ba 3 heme composition. In contrast to the previously described non‐functional quinol oxidase species with a b ‐type heme in the binuclear center [3, 4, 9, 10]this enzyme retains its electron transfer competence at wild‐type level.…”

Section: Introductioncontrasting

confidence: 74%

Expression of the Escherichia coli cyo operon in Paracoccus denitrificans results in a fully active quinol oxidase of unexpected heme composition

et al. 1998

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The cyo operon coding for the membrane-bound bo Qtype quinol oxidase of Escherichia coli has been expressed in a Paracoccus denitrificans strain deleted in its endogenous ba Q quinol oxidase. Using the P. denitrificans qox promoter, the His tagged protein complex is synthesized to a level comparable to that in E. coli and the enzyme purified in a single step on a metalchelating column. Whereas the activity of the isolated complex matches that of the oxidase purified directly from E. coli, the heterologously expressed oxidase does not show the characteristic heme composition but now carries heme a in its binuclear site.z 1998 Federation of European Biochemical Societies.

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Section: Introductioncontrasting

confidence: 74%

Expression of the Escherichia coli cyo operon in Paracoccus denitrificans results in a fully active quinol oxidase of unexpected heme composition

et al. 1998

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“…Many reports have suggested that the promiscuous assembly of terminal oxidases may be a general phenomenon in bacteria, promoted by different culture conditions or as a consequence of specific mutations (2)(3)(4)(5)(6)(7)(8)(9)(10). In most cases, hemes O and A replace each other in the high spin site of the binuclear center, producing functional enzymes with somewhat different kinetic properties (2,4,18).…”

Section: Discussionmentioning

confidence: 99%

“…Moreover, the substitution of heme O by heme B in the binuclear O 2 -reducing site in E. coli cyoE-deleted strains (cyoE encodes for farnesyl transferase that converts heme B to heme O) results in an inactive cytochrome bb 3 enzyme (10). Similarly Zikermann et al (7) reported that in Paracoccus denitrificans ctaB-deleted strains (the ctaB gene is an orthologue of cyoE), a bb 3 variant of bo 3 oxidase is enzymatically inactive. Examples of heme substitutions in the high spin heme site producing functional enzymes have been reported in several bacterial species; for example a novel b(o/a) 3 cytochrome c oxidase was purified from Bacillus stearothermophilus K17 mutants defective in caa 3 -type oxidase (8).…”

mentioning

confidence: 89%

A Novel Double Heme Substitution Produces a Functional bo 3 Variant of the Quinol Oxidase aa 3 of Bacillus cereus

Contreras-Zentella

Mendoza

Membrillo-Hernández

et al. 2003

Journal of Biological Chemistry

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A novel bo 3 -type quinol oxidase was highly purified from Bacillus cereus PYM1, a spontaneous mutant unable to synthesize heme A and therefore spectroscopically detectable cytochromes aa 3 and caa 3 . The purified enzyme contained 12.4 nmol of heme O and 11.5 nmol of heme B mg ؊1 protein. The enzyme was composed of two subunits with an M r of 51,000 and 30,000, respectively. Both subunits were immunoreactive to antibodies raised against the B cereus aa 3 oxidase. Moreover, amino-terminal sequence analysis of the 30-kDa subunit revealed that the first 19 residues were identical to those from the 30-kDa subunit of the B. cereus aa 3 oxidase. The purified bo 3 oxidase failed to oxidize ferrrocytochrome c (neither yeast nor horse) but oxidized tetrachlorohydroquinol with an apparent K m of 498 M, a V max of 21 mol of O 2 min ؊1 mg ؊1 , and a calculated turnover of 55 s ؊1 . The quinol oxidase activity with tetrachlorohydroquinol was inhibited by potassium cyanide and 2-n-heptyl 4-hydroxyquinoline-N-oxide with an I 50 of 24 and 300 M, respectively. Our results demonstrate that the bo 3 oxidase of this mutant is not the product of a new operon but instead is a cytochrome aa 3 apoprotein encoded by the qox operon of the aa 3 oxidase of B. cereus wild type promiscuously assembled with hemes B and O replacing heme A, producing a novel bo 3 cytochrome. This is the first reported example of an enzymatically active promiscuous oxidase resulting from the simultaneous substitution of its original hemes in the high and low spin sites.Bacteria have exploited unique terminal oxidases depending on the natural habitats and modes of aerobic metabolism (1). In most cases, there is more than one terminal oxidase, so that the respiratory systems of bacteria are branched. According to the nature of the electron donor two types of terminal oxidases can readily be distinguished: the cytochrome c oxidases and the quinol oxidases (1).Additionally, terminal oxidases contain different heme prosthetic groups that have provided a customary way of identification (i.e. aa 3 , bo 3 , caa 3 , bd, cbb 3 , ba 3 ). However this classification is further complicated by the fact that under specific culture conditions (2-5) or as result of mutations (6 -10), bacterial oxidases may be assembled promiscuously, accepting a different heme group to that present in its original structure. A single heme substitution (O replacing B) has been reported in the low spin heme site of cytochrome bo 3 of over-expressing strains of Escherichia coli, resulting in the assembly of a functional cytochrome, oo 3 . It is noteworthy that the heme of the binuclear O 2 reduction site was invariably heme O (6). Moreover, the substitution of heme O by heme B in the binuclear O 2 -reducing site in E. coli cyoE-deleted strains (cyoE encodes for farnesyl transferase that converts heme B to heme O) results in an inactive cytochrome bb 3 enzyme (10 (16)). In none of these mutants were any type a cytochromes spectroscopically detected; instead, the presence of a functional type o oxi...

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“…For example, a bb 3 variant of ba 3 -QOX from Paracoccus denitrificans (although enzymatically inactive) has been described (73) in which heme B substitutes for heme A in the binuclear center as a result of impaired heme A biosynthesis. It is thus possible that the novel B. subtilis oxidase corresponds to the CydAB polypeptides containing three hemes B: one low spin (b 558 ) and two high spin (b 595 and bЈ).…”

Section: A Novel Oxidase Of Bbј Type In B Subtilis Strain 168 -Thismentioning

confidence: 99%

A Cytochrome bb′-type Quinol Oxidase inBacillus subtilis Strain 168

Azarkina¹,

Siletsky²,

Borisov³

et al. 1999

Journal of Biological Chemistry

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The aerobic respiratory system of Bacillus subtilis 168 is known to contain three terminal oxidases: cytochrome caa 3 , which is a cytochrome c oxidase, and cytochrome aa 3 and bd, which are quinol oxidases. The presence of a possible fourth oxidase in the bacterium was investigated using a constructed mutant, LUH27, that lacks the aa 3 and caa 3 terminal oxidases and is also deficient in succinate:menaquinone oxidoreductase. The cytochrome bd content of LUH27 can be varied by using different growth conditions. LUH27 membranes virtually devoid of cytochrome bd respired with NADH or exogenous quinol as actively as preparations containing 0.4 nmol of cytochrome bd/mg of protein but were more sensitive to cyanide and aurachin D. The reduced minus oxidized difference spectra of the bd-deficient membranes as well as absorption changes induced by CO and cyanide indicated the presence of a "cytochrome o"-like component; however, the membranes did not contain heme O. The results provide strong evidence for the presence of a terminal oxidase of the bb type in B. subtilis. The enzyme does not pump protons and combines with CO much faster than typical heme-copper oxidases; in these respects, it resembles a cytochrome bd rather than members of the heme-copper oxidase superfamily. The genome sequence of B. subtilis 168 contains gene clusters for four respiratory oxidases. Two of these clusters, cta and qox, are deleted in LUH27. The remaining two, cydAB and ythAB, encode the identified cytochrome bd and a putative second cytochrome bd, respectively. Deletion of ythAB in strain LUH27 or the presence of the yth genes on plasmid did not affect the expression of the bb oxidase. It is concluded that the novel bb-type oxidase probably is cytochrome bd encoded by the cyd locus but with heme D being substituted by high spin heme B at the oxygen reactive site, i.e. cytochrome b 558 b 595 b.

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Expression Studies on the ba₃ Quinol Oxidase from Paracoccus Denitrificans A bb₃ Variant is Enzymatically Inactive

Cited by 19 publications

References 36 publications

Expression of the Escherichia coli cyo operon in Paracoccus denitrificans results in a fully active quinol oxidase of unexpected heme composition

Expression of the Escherichia coli cyo operon in Paracoccus denitrificans results in a fully active quinol oxidase of unexpected heme composition

A Novel Double Heme Substitution Produces a Functional bo 3 Variant of the Quinol Oxidase aa 3 of Bacillus cereus

A Cytochrome bb′-type Quinol Oxidase inBacillus subtilis Strain 168

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