Inhibition of membrane‐bound cytochrome <i>c</i> oxidase by zinc ions: High‐affinity Zn<sup>2+</sup>‐binding site at the P‐side of the membrane

Vygodina, Tatiana V.; Zakirzianova, Wiolanta; Konstantinov, Alexander A.

doi:10.1016/j.febslet.2008.11.018

Cited by 22 publications

(10 citation statements)

References 28 publications

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“…Results from earlier studies have shown that the activity of wild-type CytcO is inhibited by zinc ions (Zn 2+ ) (28)(29)(30)(31)(32)(33)(34)(35). One of the suggested Zn 2+ binding sites is located near Asp-132, consistent with structural data of the mitochondrial CytcO (36).…”

Section: Resultssupporting

confidence: 63%

“…Because proton uptake through the D pathway presumably is not rate limiting for this activity, any effects of Zn 2+ addition may reflect binding to other sites (32)(33)(34). We observed the same decrease in activity upon addition of Zn 2+ to the N139T/D132N as to the wildtype CytcO, which indicates that zinc binding to the other sites was not affected by the D pathway mutations.…”

Section: Resultssupporting

confidence: 51%

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Role of aspartate 132 at the orifice of a proton pathway in cytochromecoxidase

Johansson

Högbom

Carlsson

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Proton transfer across biological membranes underpins central processes in biological systems, such as energy conservation and transport of ions and molecules. In the membrane proteins involved in these processes, proton transfer takes place through specific pathways connecting the two sides of the membrane via control elements within the protein. It is commonly believed that acidic residues are required near the orifice of such proton pathways to facilitate proton uptake. In cytochrome c oxidase, one such pathway starts near a conserved Asp-132 residue. Results from earlier studies have shown that replacement of Asp-132 by, e.g., Asn, slows proton uptake by a factor of ∼5,000. Here, we show that proton uptake at full speed (∼10 4 s −1 ) can be restored in the Asp-132–Asn oxidase upon introduction of a second structural modification further inside the pathway (Asn-139–Thr) without compensating for the loss of the negative charge. This proton-uptake rate was insensitive to Zn 2+ addition, which in the wild-type cytochrome c oxidase slows the reaction, indicating that Asp-132 is required for Zn 2+ binding. Furthermore, in the absence of Asp-132 and with Thr at position 139, at high pH (>9), proton uptake was significantly accelerated. Thus, the data indicate that Asp-132 is not strictly required for maintaining rapid proton uptake. Furthermore, despite the rapid proton uptake in the Asn-139–Thr/Asp-132–Asn mutant cytochrome c oxidase, proton pumping was impaired, which indicates that the segment around these residues is functionally linked to pumping.

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Section: Resultssupporting

confidence: 63%

Section: Resultssupporting

confidence: 51%

Role of aspartate 132 at the orifice of a proton pathway in cytochromecoxidase

Johansson

Högbom

Carlsson

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…In fact, incorporation of CcO into liposomes was successfully used for the examination of proton pumping mechanism, ligand binding, etc. [8,9,10]. However, the liposomal turbidity does not permit the use of some of the experimental techniques, such as absorbance spectroscopy or circular dichroism.…”

Section: Introductionmentioning

confidence: 99%

Functional and structural evaluation of bovine heart cytochrome c oxidase incorporated into bicelles

et al. 2016

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Bilayered long- and short-chain phospholipid assemblies, known as bicelles, have been widely used as model membranes in biological studies. However, to date, there has been no demonstration of structural or functional viability for the fundamental mitochondrial electron transport complexes reconstituted into or interacting with bicelles. In the present work, bicelles were formed from the mixture of long- and short-chain phospholipids, specifically 14:0 and 6:0 phosphatidylcholines (1,2-dimyristoyl-sn-glycero-3-phosphocholine, (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine, (DHPC)). Isolated from bovine heart, cytochrome c oxidase was successfully incorporated into bicelles. Bicelles and cytochrome c oxidase incorporated into bicelles (“proteobicelles”) were characterized by absorption spectroscopy, dynamic light scattering, atomic force microscopy, sedimentation velocity and differential scanning calorimetry. It was demonstrated that at total concentration of phospholipids CL = 24 mM and the molar ratio (q) of long-chain DMPC over short-chain DHPC equal to 0.4, the diameter of bicelles formed at neutral pH is in the range of 30 – 60 nm with the thickness of bicelles of about 4 nm. Adding cytochrome c oxidase to bicelles unified the size of the resulting proteobicelles to about 160 nm. Cytochrome c oxidase in bicelles was fully reducible by artificial donors of electrons, exhibited “normal” reaction with external ligands, and was fully active. Both, sedimentation velocity analysis and temperature-induced denaturation indicated that enzyme in bicelles is monomeric. We concluded that cytochrome c oxidase in bicelles maintains its structural and functional integrity, and that bicelles can be used for more comprehensive investigation of cytochrome c oxidase and most likely other mitochondrial electron transfer complexes.

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“…Binding of Zn 2+ with the exit proton pathway (from the P‐side of the membrane) in the oxidized CcO required either very long preincubation with μM concentrations of Zn 2+ and the enzyme turning over, or ∼mM concentrations of Zn 2+ [19,20]. Vygodina et al [21] reported that the inhibition of the exit pathway may occur rapidly with Ki of about 1 μM, provided some high potential non‐heme center (e.g. CuB) is reduced prior to onset of the reaction with oxygen.…”

Section: Introductionmentioning

confidence: 99%

Electrochemistry suggests proton access from the exit site to the binuclear center in Paracoccus denitrificans cytochrome c oxidase pathway variants

Meyer

Melin

Richter³

et al. 2015

FEBS Letters

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a b s t r a c tTwo different pathways through which protons access cytochrome c oxidase operate during oxygen reduction from the mitochondrial matrix, or the bacterial cytoplasm. Here, we use electrocatalytic current measurements to follow oxygen reduction coupled to proton uptake in cytochrome c oxidase isolated from Paracoccus denitrificans. Wild type enzyme and site-specific variants with defects in both proton uptake pathways (K354M, D124N and K354M/D124N) were immobilized on gold nanoparticles, and oxygen reduction was probed electrochemically in the presence of varying concentrations of Zn 2+ ions, which are known to inhibit both the entry and the exit proton pathways in the enzyme. Our data suggest that under these conditions substrate protons gain access to the oxygen reduction site via the exit pathway.

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Inhibition of membrane‐bound cytochrome c oxidase by zinc ions: High‐affinity Zn²⁺‐binding site at the P‐side of the membrane

Cited by 22 publications

References 28 publications

Role of aspartate 132 at the orifice of a proton pathway in cytochromecoxidase

Role of aspartate 132 at the orifice of a proton pathway in cytochromecoxidase

Functional and structural evaluation of bovine heart cytochrome c oxidase incorporated into bicelles

Electrochemistry suggests proton access from the exit site to the binuclear center in Paracoccus denitrificans cytochrome c oxidase pathway variants

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Inhibition of membrane‐bound cytochrome c oxidase by zinc ions: High‐affinity Zn2+‐binding site at the P‐side of the membrane

Cited by 22 publications

References 28 publications

Role of aspartate 132 at the orifice of a proton pathway in cytochromecoxidase

Role of aspartate 132 at the orifice of a proton pathway in cytochromecoxidase

Functional and structural evaluation of bovine heart cytochrome c oxidase incorporated into bicelles

Electrochemistry suggests proton access from the exit site to the binuclear center in Paracoccus denitrificans cytochrome c oxidase pathway variants

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Inhibition of membrane‐bound cytochrome c oxidase by zinc ions: High‐affinity Zn²⁺‐binding site at the P‐side of the membrane