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Uncompetitive Substrate Inhibition and Noncompetitive Inhibition by 5-n-Undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) and 2-n-Nonyl-4-hydroxyquinoline-N-oxide (NQNO) is Observed for the Cytochrome bo_3Complex: Implications for a Q(H_2)-Loop Proton Translocation Mechanism

Musser, Siegfried M. and Stowell, Michael H. B. and Lee, Hung Kay and Rumbley, Jon N. and Chan, Sunney I. (1997) Uncompetitive Substrate Inhibition and Noncompetitive Inhibition by 5-n-Undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) and 2-n-Nonyl-4-hydroxyquinoline-N-oxide (NQNO) is Observed for the Cytochrome bo_3Complex: Implications for a Q(H_2)-Loop Proton Translocation Mechanism. Biochemistry, 36 (4). pp. 894-902. ISSN 0006-2960. https://resolver.caltech.edu/CaltechAUTHORS:20180212-130429431

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Abstract

The cytochrome bo3 ubiquinol oxidase complex from Escherichia coli contains two binding sites for ubiquinone(ol) (UQ(H_2)). One of these binding sites, the ubiquinol oxidation site, is clearly in dynamic equilibrium with the UQ(H_2) pool in the membrane. The second site has a high affinity for ubiquinone (UQ), stabilizes a semiquinone species, and is located physically close to the low-spin heme b component of the enzyme. The UQ molecule in this site has been proposed to remain strongly bound to the enzyme during enzyme turnover and to act as a cofactor facilitating the transfer of electrons from the substrate ubiquinol to heme b [Sato-Watanabe et al. (1994) J. Biol. Chem. 269, 28908−28912]. In this paper, the steady-state turnover of the enzyme is examined in the presence and absence of inhibitors (UHDBT and NQNO) that appear to be recognized as ubisemiquinone analogs. It is found that the kinetics are accounted for best by a noncompetitive inhibitor binding model. Furthermore, at high concentrations, the substrates ubiquinol-1 and ubiquinol-2 inhibit turnover in an uncompetitive fashion. Together, these observations strongly suggest that there must be at least two UQ(H_2) binding sites that are in rapid equilibrium with the UQ(H_2) pool under turnover conditions. Although these data do not rule out the possibility that a strongly bound UQ molecule functions to facilitate electron transfer to heme b, they are more consistent with the behavior expected if the two UQ(H_2) binding sites were to function in a Q(H_2)-loop mechanism (similar to that of the cytochrome bc1 complex) as originally proposed by Musser and co-workers [(1993) FEBS Lett. 327, 131−136]. In this model, ubiquinol is oxidized at one site and ubiquinone is reduced at the second site. While the structural similarities of the heme-copper ubiquinol and cytochrome c oxidase complexes suggest the possibility that these two families of enzymes translocate protons by similar mechanisms, the current observations indicate that the Q(H_2)-loop proton translocation mechanism for the heme-copper ubiquinol oxidase complexes should be further investigated and experimentally tested.


Item Type:Article
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http://dx.doi.org/10.1021/bi961723rDOIArticle
https://pubs.acs.org/doi/10.1021/bi961723rPublisherArticle
ORCID:
AuthorORCID
Chan, Sunney I.0000-0002-5348-2723
Additional Information:© 1997 American Chemical Society. Received 15 July 1996. Published online 28 January 1997. This work was supported by Grant GM22432 (S.I.C.) from the U.S. Public Health Service and by Grant DE-FG-02-87ER13716 (to Robert Gennis) from the U.S. Department of Energy. S.M.M. is the recipient of a National Research Service Predoctoral Award. Special thanks to Mårten Wikström (Helsinki) for sharing manuscripts before publication. Also, we thank Robert B. Gennis (University of Illinois, Urbana) for his very helpful critical readings of this manuscript.
Funders:
Funding AgencyGrant Number
NIHGM22432
Department of Energy (DOE)DE-FG-02-87ER13716
NIH Predoctoral FellowshipUNSPECIFIED
Issue or Number:4
Record Number:CaltechAUTHORS:20180212-130429431
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20180212-130429431
Official Citation:Uncompetitive Substrate Inhibition and Noncompetitive Inhibition by 5-n-Undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) and 2-n-Nonyl-4-hydroxyquinoline-N-oxide (NQNO) is Observed for the Cytochrome bo3 Complex:  Implications for a Q(H2)-Loop Proton Translocation Mechanism Siegfried M. Musser, Michael H. B. Stowell, Hung Kay Lee, Jon N. Rumbley, and Sunney I. Chan Biochemistry 1997 36 (4), 894-902 DOI: 10.1021/bi961723r
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:84791
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:13 Feb 2018 21:14
Last Modified:03 Oct 2019 19:22

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