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

Azarkina, Natalia V.; Siletsky, Sergey A.; Borisov, Vitaliy B.; Wachenfeldt, Claes von; Hederstedt, Lars; Konstantinov, Alexander A.

doi:10.1074/jbc.274.46.32810

Cited by 61 publications

(38 citation statements)

References 72 publications

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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%

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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Section: Discussionmentioning

confidence: 99%

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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“…Laser flash-induced CO photodissociation/recombination time-resolved measurements at room temperature were done as described (34). Deconvolution of the kinetic curves of CO recombination with cytochrome bd at room temperature was done with the software package GIM (subroutine "discrete") developed by A. L. Drachev in the A. N. Belozersky Institute of Physico-Chemical Biology (Moscow).…”

Section: Methodsmentioning

confidence: 99%

Interaction of Cytochrome bd with Carbon Monoxide at Low and Room Temperatures

Borisov

Sedelnikova

Poole

et al. 2001

Journal of Biological Chemistry

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Azotobacter vinelandii is an obligately aerobic bacterium in which aerotolerant dinitrogen fixation requires cytochrome bd. This oxidase comprises two polypeptide subunits and three hemes, but no copper, and has been studied extensively. However, there remain apparently conflicting reports on the reactivity of the high spin heme b 595 with ligands. Using purified cytochrome bd, we show that absorption changes induced by CO photodissociation from the fully reduced cytochrome bd at low temperatures demonstrate binding of the ligand with heme b 595 . However, the magnitude of these changes corresponds to the reaction with CO of only about 5% of the heme. CO binding with a minor fraction of heme b 595 is also revealed at room temperature by time-resolved studies of CO recombination. The data resolve the apparent discrepancies between conclusions drawn from room and low temperature spectroscopic studies of the CO reaction with cytochrome bd. The results are consistent with the proposal that hemes b 595 and d form a diheme oxygen-reducing center with a binding capacity for a single exogenous ligand molecule that partitions between the hemes d and b 595 in accordance with their intrinsic affinities for the ligand. In this model, the affinity of heme b 595 for CO is about 20-fold lower than that of heme d.Cytochrome bd is a terminal oxidase present in the respiratory chains of many bacteria (reviewed in Ref. 1, see an evolutionary tree in Ref. 2). The enzyme catalyzes reduction of molecular oxygen to water by ubiquinol or menaquinol as natural electron donors (3-5) but shows no sequence homology to the heme-copper quinol oxidases. Also, in contrast to these oxidases, cytochrome bd does not contain copper and, although generating ⌬ (6 -9), does not pump protons (10).Cytochrome bd-type oxidases purified from Azotobacter vinelandii or Escherichia coli consist of two subunits and carry three iron-porphyrin groups: low spin heme b 558 , high spin heme b 595 , and a chlorin-type high spin iron-porphyrin group (heme d). All three redox centers are proposed to be located near the periplasmic side of the membrane (2). Heme b 558 is directly involved in ubiquinol oxidation (11,12). Heme d binds O 2 and is involved in the trapping and reduction of oxygen (3). The specific role of heme b 595 is still a matter of debate. One obvious possible role is the transferrence of electrons from heme b 558 to heme d (13-15). However, because heme b 595 is high spin, it is tempting to consider its involvement in dioxygen reduction; it was proposed that hemes b 595 and d might form a binuclear dioxygen reduction center analogous to the hemecopper oxygen-reducing site in the cytochrome aa 3 -or bo 3 -type oxidases (16 -19). In such a case, one might expect heme b 595 to react with other exogenous ligands such as CO or NO as is typical of most high spin hemoproteins. Surprisingly, despite a long history of such studies, this essential and apparently simple question has not been answered by the apparently conflicting data. The intricate line shape ...

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“…To investigate this hypothesis, we tested the effects of mutations DctaCDEF, DqcrABC, and DqoxABCD in the genes for cytochrome caa 3 , cytochrome bc, and cytochrome aa 3 , respectively (van der Oost et al 1991;Azarkina et al 1999). Single deletions of ctaCDEF and qcrABC showed somewhat decreased expression of sinI-lacZ in an irondependent manner (Fig.…”

Section: Kinb Depends On and Is In A Complex With Cytochromesmentioning

confidence: 99%

Respiration control of multicellularity in Bacillus subtilis by a complex of the cytochrome chain with a membrane-embedded histidine kinase

Kolodkin‐Gal¹,

Elsholz²,

Muth³

et al. 2013

Genes Dev.

135

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Bacillussubtilis forms organized multicellular communities known as biofilms wherein the individual cells are held together by a self-produced extracellular matrix. The environmental signals that promote matrix synthesis remain largely unknown. We discovered that one such signal is impaired respiration. Specifically, high oxygen levels suppressed synthesis of the extracellular matrix. In contrast, low oxygen levels, in the absence of an alternative electron acceptor, led to increased matrix production. The response to impaired respiration was blocked in a mutant lacking cytochromes caa3 and bc and markedly reduced in a mutant lacking kinase KinB. Mass spectrometry of proteins associated with KinB showed that the kinase was in a complex with multiple components of the aerobic respiratory chain. We propose that KinB is activated via a redox switch involving interaction of its second transmembrane segment with one or more cytochromes under conditions of reduced electron transport. In addition, a second kinase (KinA) contributes to the response to impaired respiration. Evidence suggests that KinA is activated by a decrease in the nicotinamide adenine dinucleotide (NAD+)/NADH ratio via binding of NAD+ to the kinase in a PAS domain A-dependent manner. Thus, B. subtilis switches from a unicellular to a multicellular state by two pathways that independently respond to conditions of impaired respiration.

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

Cited by 61 publications

References 72 publications

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

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

Interaction of Cytochrome bd with Carbon Monoxide at Low and Room Temperatures

Respiration control of multicellularity in Bacillus subtilis by a complex of the cytochrome chain with a membrane-embedded histidine kinase

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