Electron Transfer Processes in Subunit I Mutants of Cytochrome<i>bo</i>Quinol Oxidase in<i>Escherichia coli</i>

Kobayashi, Kazuo; Tagawa, Seiichi; Mogi, Tatsushi

doi:10.1271/bbb.90105

Cited by 6 publications

(8 citation statements)

References 39 publications

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“…Furthermore, we applied pulse radiolysis to E. coli cytochrome bo mutants to elucidate the role of conserved charged amino acid residues in the subunit I mutants (Table 2). 106,107 We substituted Asp135 and Glu286 in the D-channel, Tyr288 and Lys362 in the K-channel, Arg257 and Asp407 on the periplasmic surface, and Glu540 at the cytoplasmic surface of subunit I. In all mutants, the hemes were found to be reduced in two phases.…”

Section: Chemical Reviewsmentioning

confidence: 99%

Pulse Radiolysis Studies for Mechanism in Biochemical Redox Reactions

Kobayashi

2019

Chem. Rev.

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Pulse radiolysis is a powerful method for generating highly reduced or oxidized species and free radicals. Combined with fast time-resolved spectroscopic measurement, we can monitor the reactions of intermediate species on time scales ranging from picoseconds to seconds. The application of pulse radiolysis to water generates hydrated electrons (eaq –) and specific radicals, rendering this technique useful for investigating a number of biological redox processes. The first pulse radiolysis redox investigations explored in this review involved intramolecular electron transfer processes in protein with multiple electron-accepting sites. Pulse radiolysis enabled direct monitoring of the internal electron transfer rates and the distribution of electrons within proteins. Structural information from X-ray data has allowed analysis of the rate constants and their activation parameters in relation to the mechanisms with current theoretical treatments. The second set of pulse radiolysis redox investigations explored here concerned the intermediates of enzyme reactions after redox reactions. Pulse radiolysis allowed the extremely rapid donation of electrons to a redox center in a protein. It makes it possible to observe the unstable intermediates after the reduction and the following subsequent steps. For example, the intermediates generated through the one-electron reduction of oxygenated hemoproteins, such as cytochrome P450 and nitric oxide synthase, were characterized. Interestingly, ligand exchange can occur upon the reduction of heme iron, in which different amino acid residues bind to heme in the ferrous and ferric states, respectively. We directly observed the ligand-switching intermediates of bacterial CooA, a CO sensor, and bacterial iron response regulator protein. These ligand exchange processes are physiologically important for regulating the electrode potential and effective formation of superoxide anion or HO•. The third set of pulse radiolysis redox investigations explored in this review concerns free-radical processes in biological systems. Free radicals are produced in cells and organisms in a variety of processes. The cell has developed special and very effective machinery for controlling and detoxifying reactive radicals. Radiation-generated radicals allow studies of the reactions between specific radicals and solutes, often revealing the mechanisms underlying the initial and subsequent reactions. The crucial contribution was made using pulse radiolysis techniques and knowledge of the identities, properties, and reactions of radicals. These radicals include superoxide (O2 •–), nitric monoxide (NO•), ascorbate, urate, and protein radicals. This review focuses on the reactions of these radicals and their physiological functions.

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Section: Chemical Reviewsmentioning

confidence: 99%

Pulse Radiolysis Studies for Mechanism in Biochemical Redox Reactions

Kobayashi

2019

Chem. Rev.

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“…The most intensively studied heme-copper quinol oxidase is the cytochrome bo 3 ubiquinol oxidase from Escherchia coli (cyt bo 3 ) 3 (3)(4)(5)(6)(7)(8). There are currently over 400 sequences of quinol oxidases that are homologues of cyt bo 3 .…”

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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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“…The dithionite reduced minus oxidized spectrum of the heterologously expressed vbo 3 was similar to that purified from wild type Vitreoscilla 48 and that from the ecbo 3 with the typical absorbance peaks at 428 nm, 532 nm and 563 nm 43 (Supplementary Fig. S4).…”

Section: Resultsmentioning

confidence: 60%

“…1, green sticks), is suggested to deliver 1–2 protons during reduction of the enzyme, while the D-pathway, starting at D135 in ecbo 3 (D134 in vbo 3 ) and including E286 in ecbo 3 (E285 in vbo 3 , Fig. 1 dark blue), is used for transfer of the remaining protons during the reductive and oxidative half cycles 43 . The conserved charged amino acid residues that are involved in proton uptake and/or pumping are listed in Table 1 of the Supplementary Information.…”

Section: Resultsmentioning

confidence: 99%

The proton pumping bo oxidase from Vitreoscilla

Graf

Brzezinski

Ballmoos

2019

Sci Rep

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The cytochrome bo3 quinol oxidase from Vitreoscilla (vbo3) catalyses oxidation of ubiquinol and reduction of O2 to H2O. Data from earlier studies suggested that the free energy released in this reaction is used to pump sodium ions instead of protons across a membrane. Here, we have studied the functional properties of heterologously expressed vbo3 with a variety of methods. (i) Following oxygen consumption with a Clark-type electrode, we did not observe a measurable effect of Na+ on the oxidase activity of purified vbo3 solubilized in detergent or reconstituted in liposomes. (ii) Using fluorescent dyes, we find that vbo3 does not pump Na+ ions, but H+ across the membrane, and that H+-pumping is not influenced by the presence of Na+. (iii) Using an oxygen pulse method, it was found that 2 H+/e− are ejected from proteoliposomes, in agreement with the values found for the H+-pumping bo3 oxidase of Escherichia coli (ecbo3). This coincides with the interpretation that 1 H+/e− is pumped across the membrane and 1 H+/e− is released during quinol oxidation. (iv) When the electron transfer kinetics of vbo3 upon reaction with oxygen were followed in single turnover experiments, a similar sequence of reaction steps was observed as reported for the E. coli enzyme and none of these reactions was notably affected by the presence of Na+. Overall the data show that vbo3 is a proton pumping terminal oxidase, behaving similarly to the Escherichia coli bo3 quinol oxidase.

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Electron Transfer Processes in Subunit I Mutants of CytochromeboQuinol Oxidase inEscherichia coli

Cited by 6 publications

References 39 publications

Pulse Radiolysis Studies for Mechanism in Biochemical Redox Reactions

Pulse Radiolysis Studies for Mechanism in Biochemical Redox Reactions

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

The proton pumping bo oxidase from Vitreoscilla

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