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The Maturation Factors HoxR and HoxT Contribute to Oxygen Tolerance of Membrane-Bound (NiFe) Hydrogenase in Ralstonia eutropha H16

The Maturation Factors HoxR and HoxT Contribute to Oxygen Tolerance of Membrane-Bound (NiFe)... The Maturation Factors HoxR and HoxT Contribute to Oxygen Tolerance of Membrane-Bound (NiFe) Hydrogenase in Ralstonia eutropha H16 ▿ † Johannes Fritsch , Oliver Lenz and Bärbel Friedrich * Institut für Biologie/Mikrobiologie, Humboldt-Universität zu Berlin, Chausseestrasse 117, 10115 Berlin, Germany ABSTRACT The membrane-bound (NiFe) hydrogenase (MBH) of Ralstonia eutropha H16 undergoes a complex maturation process comprising cofactor assembly and incorporation, subunit oligomerization, and finally twin-arginine-dependent membrane translocation. Due to its outstanding O 2 and CO tolerance, the MBH is of biotechnological interest and serves as a molecular model for a robust hydrogen catalyst. Adaptation of the enzyme to oxygen exposure has to take into account not only the catalytic reaction but also biosynthesis of the intricate redox cofactors. Here, we report on the role of the MBH-specific accessory proteins HoxR and HoxT, which are key components in MBH maturation at ambient O 2 levels. MBH-driven growth on H 2 is inhibited or retarded at high O 2 partial pressure (pO 2 ) in mutants inactivated in the hoxR and hoxT genes. The ratio of mature and nonmature forms of the MBH small subunit is shifted toward the precursor form in extracts derived from the mutant cells grown at high pO 2 . Lack of hoxR and hoxT can phenotypically be restored by providing O 2 -limited growth conditions. Analysis of copurified maturation intermediates leads to the conclusion that the HoxR protein is a constituent of a large transient protein complex, whereas the HoxT protein appears to function at a final stage of MBH maturation. UV-visible spectroscopy of heterodimeric MBH purified from hoxR mutant cells points to alterations of the Fe-S cluster composition. Thus, HoxR may play a role in establishing a specific Fe-S cluster profile, whereas the HoxT protein seems to be beneficial for cofactor stability under aerobic conditions. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Bacteriology American Society For Microbiology

The Maturation Factors HoxR and HoxT Contribute to Oxygen Tolerance of Membrane-Bound (NiFe) Hydrogenase in Ralstonia eutropha H16

Journal of Bacteriology , Volume 193 (10): 2487 – May 15, 2011

The Maturation Factors HoxR and HoxT Contribute to Oxygen Tolerance of Membrane-Bound (NiFe) Hydrogenase in Ralstonia eutropha H16

Journal of Bacteriology , Volume 193 (10): 2487 – May 15, 2011

Abstract

The Maturation Factors HoxR and HoxT Contribute to Oxygen Tolerance of Membrane-Bound (NiFe) Hydrogenase in Ralstonia eutropha H16 ▿ † Johannes Fritsch , Oliver Lenz and Bärbel Friedrich * Institut für Biologie/Mikrobiologie, Humboldt-Universität zu Berlin, Chausseestrasse 117, 10115 Berlin, Germany ABSTRACT The membrane-bound (NiFe) hydrogenase (MBH) of Ralstonia eutropha H16 undergoes a complex maturation process comprising cofactor assembly and incorporation, subunit oligomerization, and finally twin-arginine-dependent membrane translocation. Due to its outstanding O 2 and CO tolerance, the MBH is of biotechnological interest and serves as a molecular model for a robust hydrogen catalyst. Adaptation of the enzyme to oxygen exposure has to take into account not only the catalytic reaction but also biosynthesis of the intricate redox cofactors. Here, we report on the role of the MBH-specific accessory proteins HoxR and HoxT, which are key components in MBH maturation at ambient O 2 levels. MBH-driven growth on H 2 is inhibited or retarded at high O 2 partial pressure (pO 2 ) in mutants inactivated in the hoxR and hoxT genes. The ratio of mature and nonmature forms of the MBH small subunit is shifted toward the precursor form in extracts derived from the mutant cells grown at high pO 2 . Lack of hoxR and hoxT can phenotypically be restored by providing O 2 -limited growth conditions. Analysis of copurified maturation intermediates leads to the conclusion that the HoxR protein is a constituent of a large transient protein complex, whereas the HoxT protein appears to function at a final stage of MBH maturation. UV-visible spectroscopy of heterodimeric MBH purified from hoxR mutant cells points to alterations of the Fe-S cluster composition. Thus, HoxR may play a role in establishing a specific Fe-S cluster profile, whereas the HoxT protein seems to be beneficial for cofactor stability under aerobic conditions.

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References (89)

Publisher
American Society For Microbiology
Copyright
Copyright © 2011 by the American society for Microbiology.
ISSN
0021-9193
eISSN
1098-5530
DOI
10.1128/JB.01427-10
pmid
21441514
Publisher site
See Article on Publisher Site

Abstract

The Maturation Factors HoxR and HoxT Contribute to Oxygen Tolerance of Membrane-Bound (NiFe) Hydrogenase in Ralstonia eutropha H16 ▿ † Johannes Fritsch , Oliver Lenz and Bärbel Friedrich * Institut für Biologie/Mikrobiologie, Humboldt-Universität zu Berlin, Chausseestrasse 117, 10115 Berlin, Germany ABSTRACT The membrane-bound (NiFe) hydrogenase (MBH) of Ralstonia eutropha H16 undergoes a complex maturation process comprising cofactor assembly and incorporation, subunit oligomerization, and finally twin-arginine-dependent membrane translocation. Due to its outstanding O 2 and CO tolerance, the MBH is of biotechnological interest and serves as a molecular model for a robust hydrogen catalyst. Adaptation of the enzyme to oxygen exposure has to take into account not only the catalytic reaction but also biosynthesis of the intricate redox cofactors. Here, we report on the role of the MBH-specific accessory proteins HoxR and HoxT, which are key components in MBH maturation at ambient O 2 levels. MBH-driven growth on H 2 is inhibited or retarded at high O 2 partial pressure (pO 2 ) in mutants inactivated in the hoxR and hoxT genes. The ratio of mature and nonmature forms of the MBH small subunit is shifted toward the precursor form in extracts derived from the mutant cells grown at high pO 2 . Lack of hoxR and hoxT can phenotypically be restored by providing O 2 -limited growth conditions. Analysis of copurified maturation intermediates leads to the conclusion that the HoxR protein is a constituent of a large transient protein complex, whereas the HoxT protein appears to function at a final stage of MBH maturation. UV-visible spectroscopy of heterodimeric MBH purified from hoxR mutant cells points to alterations of the Fe-S cluster composition. Thus, HoxR may play a role in establishing a specific Fe-S cluster profile, whereas the HoxT protein seems to be beneficial for cofactor stability under aerobic conditions.

Journal

Journal of BacteriologyAmerican Society For Microbiology

Published: May 15, 2011

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