Journal
ACS CATALYSIS
Volume 7, Issue 6, Pages 3916-3923Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acscatal.7b01286
Keywords
bilirubin oxidase; chloride inhibition; pH influence; electrochemistry; catalysis; resting forms
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Funding
- Region Provence-Alpes-Cote d'Azur
- Region Aquitaine
- ANR [RATIOCELLS-ANR-12-BS08-0011-01, CAROUCELL ANR-13-BIOME-0003-02]
- Labex AMADEus [ANR-10-IDEX-003-02]
- National Institute of Diabetes and Digestive and Kidney Diseases [R01DK31450]
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Bilirubin oxidases (BODs) belong to the multicopper wddase (MCO) family and efficiently reduce 02 at neutral pH and under physiological conditions where chloride concentrations are >100 mM. BODs were consequently considered to be resistant, as opposed to laccases. However, there has not been a detailed study of the related effect of chloride and pH on the redox state of immobilized BODs. Here, we investigate by electrochemistry the catalytic mechanism of O-2 reduction by the thermostable Bacillus pumilus BOD immobilized on carbon nanofibers in the presence of NaCl. The addition of chloride results in the formation of a redox state of the enzyme, previously observed for different BODs and laccases, which is active only after a reductive step. This behavior has not been previously investigated. We show that the kinetics of formation of this state is strongly dependent on pH, temperature, Cl- concentration, and applied redox potential. Ultraviolet-visible spectroscopy allows us to correlate the inhibition by chloride with the formation of the alternative resting form of the enzyme. We demonstrate that O-2 is not required for its formation and show that the application of an oxidative potential is sufficient. In addition, our results suggest that reactivation may proceed through T3 beta.
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