Role of a bound ubiquinone on reactions of the <i>Escherichia coli</i> cytochrome <i>bo</i> with ubiquinol and dioxygen

Mogi, Tatsushi; Sato-Watanabe, Mariko; Miyoshi, Hideto; Orii, Yutaka

doi:10.1016/s0014-5793(99)01007-8

Cited by 19 publications

(16 citation statements)

References 22 publications

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“…These results provide further support for our proposal that the Cu A end of the cupredoxin fold and the quinol oxidase specific (Qox) domain provides the Q L site and is involved in electron transfer to the metal centers in subunit 1 (15). Since the bound ubiquinone at the Q H site is essential for the quinol oxidation at the Q L site (16,18,19), the Q H site may be located in transmembrane helices of subunit II and/or subunit I.…”

Section: Resultssupporting

confidence: 72%

Defining the Structural Domain of Subunit II of the Heme-Copper Terminal Oxidase Using Chimeric Enzymes Constructed from the Escherichia coli bo-Type Ubiquinol Oxidase and the Thermophilic Bacillus caa3-Type Cytochrome c Oxidase

Sakamoto¹,

Mogi

Noguchi³

et al. 1999

Journal of Biochemistry

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To probe the location of the quinol oxidation site and physical interactions for inter-subunit electron transfer, we constructed and characterized two chimeric oxidases in which subunit II (CyoA) of cytochrome bo-type ubiquinol oxidase from Escherichia coli was replaced with the counterpart (CaaA) of caa(3)-type cytochrome c oxidase from thermophilic Bacillus PS3. In pHNchi5, the C-terminal hydrophilic domain except a connecting region as to transmembrane helix II of CyoA was replaced with the counterpart of CaaA, which carries the Cu(A) site and cytochrome c domain. The resultant chimeric oxidase was detected immunochemically and spectroscopically, and the turnover numbers for Q(1)H(2) (ubiquinol-1) and TMPD (N,N, N',N'-tetramethyl-p-phenylenediamine) oxidation were 28 and 8.5 s(-1), respectively. In pHNchi6, the chimeric oxidase was designed to carry a minimal region of the cupredoxin fold containing all the Cu(A) ligands, and showed enzymatic activities of 65 and 5.1 s(-1), and an expression level better than that of pHNchi5. Kinetic analyses proved that the apparent lower turnover of the chimeric enzyme by pHNchi6 was due to the higher K(m) of the enzyme for Q(1)H(2) (220 microM) than that of cytochrome bo (48 microM), while in the enzyme by pHNchi5, both substrate-binding and internal electron transfer were perturbed. These results suggest that the connecting region and the C-terminal alpha(1)-alpha(2)-beta(11)-alpha(3) domain of CyoA are involved in the quinol oxidation and/or physical interactions for inter-subunit electron transfer, supporting our previous proposal [Sato-Watanabe, M., Mogi, T., Miyoshi, H., and Anraku, Y. (1998) Biochemistry 37, 12744-12752]. The close relationship of E. coli quinol oxidases to cytochrome c oxidase of Gram-positive bacteria like Bacillus was also indicated.

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Section: Resultssupporting

confidence: 72%

Defining the Structural Domain of Subunit II of the Heme-Copper Terminal Oxidase Using Chimeric Enzymes Constructed from the Escherichia coli bo-Type Ubiquinol Oxidase and the Thermophilic Bacillus caa3-Type Cytochrome c Oxidase

Sakamoto¹,

Mogi

Noguchi³

et al. 1999

Journal of Biochemistry

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“…The protein binding site which stabilizes the semiquinone is referred to as the high affinity Q-binding site, or Q H . Stabilizing the 1-electron reduced form of the quinone is consistent with the quinone bound at this site acting as a “pair splitter”, which can be reduced by two electrons and then transfer the electrons one-at-a-time to a 1-electron acceptor, heme b [19, 20]. The rate of formation of the semiquinone upon mixing reduced ubiquinol-2 with the enzyme has been monitored by rapid freeze-quench EPR spectroscopy, and the results indicate that the semiquinone formed at the Q H -site is an intermediate in the catalytic mechanism [21].…”

Section: Introductionmentioning

confidence: 88%

“…The semiquinone at the Q H -site is formed within 10 μs after pulsed radiolysis, and electron transfer from the semiquinone to heme b follows, with a first order rate constant of 1.5 × 10 3 s −1 [22]. Studies of the reaction of the fully reduced enzyme with O 2 have shown that electron transfer from heme b to the heme o 3 /Cu B binuclear center occurs with a rate of about 10 4 s −1 [19, 23]. A comparison of the intramolecular electron transfer events for enzyme containing the quinone bound at the Q H -site with enzyme from which the quinone has been removed (prepared in Triton X-100) also supports the model that the tightly bound reduced quinone is capable of rapid electron transfer to heme b [12].…”

Section: Introductionmentioning

confidence: 99%

The quinone-binding sites of the cytochrome bo3 ubiquinol oxidase from Escherichia coli

Yap

Lin

Ouyang

et al. 2010

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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Summary Cytochrome bo3 is the major respiratory oxidase located in the cytoplasmic membrane of E. coli when grown under high oxygen tension. The enzyme catalyzes the 2-electron oxidation of ubiquinol-8 and the 4-electron reduction of dioxygen to water. When solubilized and isolated using dodecylmaltoside, the enzyme contains one equivalent of ubiquinone-8, bound at a high affinity site (QH). The quinone bound at the QH site can form a stable semiquinone, and the amino acid residues which hydrogen bond to the semiquinone have been identified. In the current work, it is shown that the tightly bound ubiquinone-8 at the QH site is not displaced by ubiquinol-1 even during enzyme turnover. Furthermore, the presence of high affinity inhibitors, HQNO and aurachin C1-10, do not displace ubiquinone-8 from the QH site. The data clearly support the existence of a second binding site for ubiquinone, the QL site, which can rapidly exchange with the substrate pool. HQNO is shown to bind to a single site on the enzyme and to prevent formation of the stable ubisemiquinone, though without displacing the bound quinone. Inhibition of the steady state kinetics of the enzyme indicates that aurachin C1-10 may compete for binding with quinol at the QL site while, at the same time, preventing formation of the ubisemiquinone at the QH site. It is suggested that the two quinone binding sites may be adjacent to each other or partially overlap.

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“…Despite significant effort (17,19,20), little is known about the location of this site within the protein (21). Cyt bo 3 , when purified using the detergent dodecylmaltoside, has 1 eq of ubiquinol-8 bound at the Q H -site, and this bound quinol does not readily exchange with the free quinol in the membrane (18,(22)(23)(24). The ubiquinone bound at the Q H -site functions as a cofactor, accepting two electrons from the quinol at the Q L -site and passing the electrons on to heme b one at a time.…”

mentioning

confidence: 99%

Characterization of the Semiquinone Radical Stabilized by the Cytochrome aa3-600 Menaquinol Oxidase of Bacillus subtilis

Narasimhulu

Samoilova

et al. 2010

Journal of Biological Chemistry

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Cytochrome aa 3 -600 is one of the principle respiratory oxidases from Bacillus subtilis and is a member of the heme-copper superfamily of oxygen reductases. This enzyme catalyzes the two-electron oxidation of menaquinol and the four-electron reduction of O 2 to 2H 2 O. Cytochrome aa 3 -600 is of interest because it is a very close homologue of the cytochrome bo 3 ubiquinol oxidase from Escherichia coli, except that it uses menaquinol instead of ubiquinol as a substrate. One question of interest is how the proteins differ in response to the differences in structure and electrochemical properties between ubiquinol and menaquinol. Cytochrome bo 3 has a high affinity binding site for ubiquinol that stabilizes a ubi-semiquinone. This has permitted the use of pulsed EPR techniques to investigate the protein interaction with the ubiquinone. The current work initiates studies to characterize the equivalent site in cytochrome aa 3 -600. Cytochrome aa 3 -600 has been cloned and expressed in a His-tagged form in B. subtilis. After isolation of the enzyme in dodecylmaltoside, it is shown that the pure enzyme contains 1 eq of menaquinone-7 and that the enzyme stabilizes a menasemiquinone. Pulsed EPR studies have shown that there are both similarities as well as significant differences in the interactions of the mena-semiquinone with cytochrome aa 3 -600 in comparison with the ubi-semiquinone in cytochrome bo 3 . Our data indicate weaker hydrogen bonds of the menaquinone in cytochrome aa 3 -600 in comparison with ubiquinone in cytochrome bo 3 . In addition, the electronic structure of the semiquinone cyt aa 3 -600 is more shifted toward the anionic form from the neutral state in cyt bo 3 .A number of prokaryotes contain heme-copper respiratory oxygen reductases, which utilize a membrane-bound quinol as the substrate (electron donor) (1, 2). These enzymes (quinol oxidases) are closely related to the cytochrome c oxidases, reduce O 2 to water, and also pump protons across the membrane bilayer, generating a proton motive force. The quinol oxidases lack Cu A , which is present in the cytochrome c oxidases, and the amino acid sequences of the quinol oxidases can be distinguished from those of the cytochrome c oxidases by the lack of the Cu A binding motif.The most intensively studied heme-copper quinol oxidase is the cytochrome bo 3 ubiquinol oxidase from Escherchia coli (cyt bo 3 ) 3 (3-8). There are currently over 400 sequences of quinol oxidases that are homologues of cyt bo 3 . The vast majority of these sequences are from proteobacteria (330 sequences) or the firmicutes (80 sequences). Bacillus subtilis, a firmicute, does not contain ubiquinone but relies on menaquinone (see Fig. 1) as a redox component in its aerobic respiratory chain (9). There is a homologue of cyt bo 3 in B. subtilis called cytochrome aa 3 -600, and as expected, this enzyme is a menaquinol oxidase (10 -16). Whereas E. coli cyt bo 3 uses only ubiquinol as a substrate, the B. subtilis cyt aa 3 -600 is strictly a menaquinol oxidase. The motivation of the cu...

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Role of a bound ubiquinone on reactions of the Escherichia coli cytochrome bo with ubiquinol and dioxygen

Cited by 19 publications

References 22 publications

Defining the Structural Domain of Subunit II of the Heme-Copper Terminal Oxidase Using Chimeric Enzymes Constructed from the Escherichia coli bo-Type Ubiquinol Oxidase and the Thermophilic Bacillus caa3-Type Cytochrome c Oxidase

Defining the Structural Domain of Subunit II of the Heme-Copper Terminal Oxidase Using Chimeric Enzymes Constructed from the Escherichia coli bo-Type Ubiquinol Oxidase and the Thermophilic Bacillus caa3-Type Cytochrome c Oxidase

The quinone-binding sites of the cytochrome bo3 ubiquinol oxidase from Escherichia coli

Characterization of the Semiquinone Radical Stabilized by the Cytochrome aa3-600 Menaquinol Oxidase of Bacillus subtilis

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