Zinc inhibition of bacterial cytochrome bc1 reveals the role of cytochrome b E295 in proton release at the Qo site

Dong Woo Lee, Youssef El Khoury, Francesco Francia, Barbara Zambelli, Stefano Ciurli, Giovanni Venturoli, Petra Hellwig, Fevzi Daldal

Research output: Contribution to journalArticle

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Abstract

The cytochrome (cyt) bc1 complex (cyt bc1) plays a major role in the electrogenic extrusion of protons across the membrane responsible for the proton motive force to produce ATP. Proton-coupled electron transfer underlying the catalysis of cyt bc1 is generally accepted, but the molecular basis of coupling and associated proton efflux pathway(s) remains unclear. Herein we studied Zn2+-induced inhibition of Rhodobacter capsulatus cyt bc1 using enzyme kinetics, isothermal titration calorimetry (ITC), and electrochemically induced Fourier transform infrared (FTIR) difference spectroscopy with the purpose of understanding the Zn2+ binding mechanism and its inhibitory effect on cyt bc 1 function. Analogous studies were conducted with a mutant of cyt b, E295, a residue previously proposed to bind Zn2+ on the basis of extended X-ray absorption fine-structure spectroscopy. ITC analysis indicated that mutation of E295 to valine, a noncoordinating residue, results in a decrease in Zn2+ binding affinity. The kinetic study showed that wild-type cyt bc1 and its E295V mutant have similar levels of apparent Km values for decylbenzohydroquinone as a substrate (4.9 ± 0.2 and 3.1 ± 0.4 μM, respectively), whereas their K I values for Zn2+ are 8.3 and 38.5 μM, respectively. The calorimetry-based KD values for the high-affinity site of cyt bc1 are on the same order of magnitude as the KI values derived from the kinetic analysis. Furthermore, the FTIR signal of protonated acidic residues was perturbed in the presence of Zn2+, whereas the E295V mutant exhibited no significant change in electrochemically induced FTIR difference spectra measured in the presence and absence of Zn2+. Our overall results indicate that the proton-active E295 residue near the Q o site of cyt bc1 can bind directly to Zn2+, resulting in a decrease in the electron transferring activity without changing drastically the redox potentials of the cofactors of the enzyme. We conclude that E295 is involved in proton efflux coupled to electron transfer at the Qo site of cyt bc1.

Original languageEnglish
Pages (from-to)4263-4272
Number of pages10
JournalBiochemistry
Volume50
Issue number20
DOIs
Publication statusPublished - 2011 May 24

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Cytochromes b
Electron Transport Complex III
Protons
Zinc
Calorimetry
Fourier transforms
Fourier Analysis
Electrons
Infrared radiation
Titration
Rhodobacter capsulatus
Extended X ray absorption fine structure spectroscopy
Enzyme inhibition
Enzyme kinetics
Proton-Motive Force
Coenzymes
Valine
Fourier Transform Infrared Spectroscopy
Cytochromes
Catalysis

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Lee, D. W., El Khoury, Y., Francia, F., Zambelli, B., Ciurli, S., Venturoli, G., ... Daldal, F. (2011). Zinc inhibition of bacterial cytochrome bc1 reveals the role of cytochrome b E295 in proton release at the Qo site. Biochemistry, 50(20), 4263-4272. https://doi.org/10.1021/bi200230e
Lee, Dong Woo ; El Khoury, Youssef ; Francia, Francesco ; Zambelli, Barbara ; Ciurli, Stefano ; Venturoli, Giovanni ; Hellwig, Petra ; Daldal, Fevzi. / Zinc inhibition of bacterial cytochrome bc1 reveals the role of cytochrome b E295 in proton release at the Qo site. In: Biochemistry. 2011 ; Vol. 50, No. 20. pp. 4263-4272.
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abstract = "The cytochrome (cyt) bc1 complex (cyt bc1) plays a major role in the electrogenic extrusion of protons across the membrane responsible for the proton motive force to produce ATP. Proton-coupled electron transfer underlying the catalysis of cyt bc1 is generally accepted, but the molecular basis of coupling and associated proton efflux pathway(s) remains unclear. Herein we studied Zn2+-induced inhibition of Rhodobacter capsulatus cyt bc1 using enzyme kinetics, isothermal titration calorimetry (ITC), and electrochemically induced Fourier transform infrared (FTIR) difference spectroscopy with the purpose of understanding the Zn2+ binding mechanism and its inhibitory effect on cyt bc 1 function. Analogous studies were conducted with a mutant of cyt b, E295, a residue previously proposed to bind Zn2+ on the basis of extended X-ray absorption fine-structure spectroscopy. ITC analysis indicated that mutation of E295 to valine, a noncoordinating residue, results in a decrease in Zn2+ binding affinity. The kinetic study showed that wild-type cyt bc1 and its E295V mutant have similar levels of apparent Km values for decylbenzohydroquinone as a substrate (4.9 ± 0.2 and 3.1 ± 0.4 μM, respectively), whereas their K I values for Zn2+ are 8.3 and 38.5 μM, respectively. The calorimetry-based KD values for the high-affinity site of cyt bc1 are on the same order of magnitude as the KI values derived from the kinetic analysis. Furthermore, the FTIR signal of protonated acidic residues was perturbed in the presence of Zn2+, whereas the E295V mutant exhibited no significant change in electrochemically induced FTIR difference spectra measured in the presence and absence of Zn2+. Our overall results indicate that the proton-active E295 residue near the Q o site of cyt bc1 can bind directly to Zn2+, resulting in a decrease in the electron transferring activity without changing drastically the redox potentials of the cofactors of the enzyme. We conclude that E295 is involved in proton efflux coupled to electron transfer at the Qo site of cyt bc1.",
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Lee, DW, El Khoury, Y, Francia, F, Zambelli, B, Ciurli, S, Venturoli, G, Hellwig, P & Daldal, F 2011, 'Zinc inhibition of bacterial cytochrome bc1 reveals the role of cytochrome b E295 in proton release at the Qo site', Biochemistry, vol. 50, no. 20, pp. 4263-4272. https://doi.org/10.1021/bi200230e

Zinc inhibition of bacterial cytochrome bc1 reveals the role of cytochrome b E295 in proton release at the Qo site. / Lee, Dong Woo; El Khoury, Youssef; Francia, Francesco; Zambelli, Barbara; Ciurli, Stefano; Venturoli, Giovanni; Hellwig, Petra; Daldal, Fevzi.

In: Biochemistry, Vol. 50, No. 20, 24.05.2011, p. 4263-4272.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Zinc inhibition of bacterial cytochrome bc1 reveals the role of cytochrome b E295 in proton release at the Qo site

AU - Lee, Dong Woo

AU - El Khoury, Youssef

AU - Francia, Francesco

AU - Zambelli, Barbara

AU - Ciurli, Stefano

AU - Venturoli, Giovanni

AU - Hellwig, Petra

AU - Daldal, Fevzi

PY - 2011/5/24

Y1 - 2011/5/24

N2 - The cytochrome (cyt) bc1 complex (cyt bc1) plays a major role in the electrogenic extrusion of protons across the membrane responsible for the proton motive force to produce ATP. Proton-coupled electron transfer underlying the catalysis of cyt bc1 is generally accepted, but the molecular basis of coupling and associated proton efflux pathway(s) remains unclear. Herein we studied Zn2+-induced inhibition of Rhodobacter capsulatus cyt bc1 using enzyme kinetics, isothermal titration calorimetry (ITC), and electrochemically induced Fourier transform infrared (FTIR) difference spectroscopy with the purpose of understanding the Zn2+ binding mechanism and its inhibitory effect on cyt bc 1 function. Analogous studies were conducted with a mutant of cyt b, E295, a residue previously proposed to bind Zn2+ on the basis of extended X-ray absorption fine-structure spectroscopy. ITC analysis indicated that mutation of E295 to valine, a noncoordinating residue, results in a decrease in Zn2+ binding affinity. The kinetic study showed that wild-type cyt bc1 and its E295V mutant have similar levels of apparent Km values for decylbenzohydroquinone as a substrate (4.9 ± 0.2 and 3.1 ± 0.4 μM, respectively), whereas their K I values for Zn2+ are 8.3 and 38.5 μM, respectively. The calorimetry-based KD values for the high-affinity site of cyt bc1 are on the same order of magnitude as the KI values derived from the kinetic analysis. Furthermore, the FTIR signal of protonated acidic residues was perturbed in the presence of Zn2+, whereas the E295V mutant exhibited no significant change in electrochemically induced FTIR difference spectra measured in the presence and absence of Zn2+. Our overall results indicate that the proton-active E295 residue near the Q o site of cyt bc1 can bind directly to Zn2+, resulting in a decrease in the electron transferring activity without changing drastically the redox potentials of the cofactors of the enzyme. We conclude that E295 is involved in proton efflux coupled to electron transfer at the Qo site of cyt bc1.

AB - The cytochrome (cyt) bc1 complex (cyt bc1) plays a major role in the electrogenic extrusion of protons across the membrane responsible for the proton motive force to produce ATP. Proton-coupled electron transfer underlying the catalysis of cyt bc1 is generally accepted, but the molecular basis of coupling and associated proton efflux pathway(s) remains unclear. Herein we studied Zn2+-induced inhibition of Rhodobacter capsulatus cyt bc1 using enzyme kinetics, isothermal titration calorimetry (ITC), and electrochemically induced Fourier transform infrared (FTIR) difference spectroscopy with the purpose of understanding the Zn2+ binding mechanism and its inhibitory effect on cyt bc 1 function. Analogous studies were conducted with a mutant of cyt b, E295, a residue previously proposed to bind Zn2+ on the basis of extended X-ray absorption fine-structure spectroscopy. ITC analysis indicated that mutation of E295 to valine, a noncoordinating residue, results in a decrease in Zn2+ binding affinity. The kinetic study showed that wild-type cyt bc1 and its E295V mutant have similar levels of apparent Km values for decylbenzohydroquinone as a substrate (4.9 ± 0.2 and 3.1 ± 0.4 μM, respectively), whereas their K I values for Zn2+ are 8.3 and 38.5 μM, respectively. The calorimetry-based KD values for the high-affinity site of cyt bc1 are on the same order of magnitude as the KI values derived from the kinetic analysis. Furthermore, the FTIR signal of protonated acidic residues was perturbed in the presence of Zn2+, whereas the E295V mutant exhibited no significant change in electrochemically induced FTIR difference spectra measured in the presence and absence of Zn2+. Our overall results indicate that the proton-active E295 residue near the Q o site of cyt bc1 can bind directly to Zn2+, resulting in a decrease in the electron transferring activity without changing drastically the redox potentials of the cofactors of the enzyme. We conclude that E295 is involved in proton efflux coupled to electron transfer at the Qo site of cyt bc1.

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