Features and functional importance of key residues of the Mycobacterium tuberculosis cytochrome bd oxidase
Cytochrome bd (cyt-bd) oxygen reductases have a high affinity to oxygen and use the two electrons provided by ubiquinol or menaquinol, like in mycobacteria, to reduce oxygen to water. Although they do not pump protons from the cytoplasmic to the periplasmic side, they generate a proton motive fo...
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Main Authors: | , , , |
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Format: | Article |
Language: | English |
Published: |
2021
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Subjects: | |
Online Access: | https://hdl.handle.net/10356/149230 |
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Institution: | Nanyang Technological University |
Language: | English |
Summary: | Cytochrome bd (cyt-bd) oxygen reductases have a
high affinity to oxygen and use the two electrons provided by
ubiquinol or menaquinol, like in mycobacteria, to reduce oxygen to
water. Although they do not pump protons from the cytoplasmic
to the periplasmic side, they generate a proton motive force due to
the release of protons after quinol oxidation. Here, we show that
the mycobacterial cyt-bd has a number of specific features,
including a 17-residue stretch (307SGVTLQGIRDLQQEYQQ323)
near the Q-loop of the Mycobacterium tuberculosis subunit CydA
and a 412QLVRLTVKA420 region on the periplasmic side. Site
directed mutagenesis and whole-bacteria assays demonstrated that
these mycobacteria-specific stretches are essential for the oxidase’s function. Single amino acid substitutions around the
307SGVTLQGIRDLQQEYQQ323 stretch revealed the importance of the aromatic residue Y330 in oxygen consumption and
consequently in ATP synthesis. A moderate reduction and no effect was observed for mutants F325 and Y321, respectively, while the
double mutant CydAY321/F325 drastically reduced enzyme activity. In addition, single mutants of the mycobacterial cyt-bd were
generated to probe the role of proposed critical residues for proton shuffling. Further data demonstrate that amino acids W64 and
F18 in the CydB subunit might be important as any slight destabilization of the hydrophobic environment near them makes the
enzyme inactive. Finally, the potential of the mycobacterial cyt-bd as a drug target is discussed. |
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