C-Terminal Truncation and Histidine-Tagging of Cytochrome <i>c</i> Oxidase Subunit II Reveals the Native Processing Site, Shows Involvement of the C-Terminus in Cytochrome <i>c</i> Binding, and Improves the Assay for Proton Pumping

Hiser, Carrie; Mills, Denise A.; Schall, Michael; Ferguson‐Miller, Shelagh

doi:10.1021/bi0018988

Cited by 38 publications

(59 citation statements)

References 36 publications

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“…To maximize the homogeneity of the protein preparation, we took two approaches. First, a 6-histidine tag was attached to the C terminus of an artificially truncated subunit II (13) (Fig. 1), providing a uniform subunit II peptide.…”

Section: Resultsmentioning

confidence: 99%

Identification of conserved lipid/detergent-binding sites in a high-resolution structure of the membrane protein cytochrome c oxidase

Qin

Hiser

Mulichak

et al. 2006

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

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284

385

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Well ordered reproducible crystals of cytochrome c oxidase (CcO) from Rhodobacter sphaeroides yield a previously unreported structure at 2.0 Å resolution that contains the two catalytic subunits and a number of alkyl chains of lipids and detergents. Comparison with crystal structures of other bacterial and mammalian CcOs reveals that the positions occupied by native membrane lipids and detergent substitutes are highly conserved, along with amino acid residues in their vicinity, suggesting a more prevalent and specific role of lipid in membrane protein structure than often envisioned. Well defined detergent head groups (maltose) are found associated with aromatic residues in a manner similar to phospholipid head groups, likely contributing to the success of alkyl glycoside detergents in supporting membrane protein activity and crystallizability. Other significant features of this structure include the following: finding of a previously unreported crystal contact mediated by cadmium and an engineered histidine tag; documentation of the unique His-Tyr covalent linkage close to the active site; remarkable conservation of a chain of waters in one proton pathway (D-path); and discovery of an inhibitory cadmium-binding site at the entrance to another proton path (K-path). These observations provide important insight into CcO structure and mechanism, as well as the significance of bound lipid in membrane proteins.cadmium binding ͉ membrane protein structure ͉ proton-conducting water chains C ytochrome c oxidase (CcO) is the terminal enzyme in the electron transfer chain in eukaryotes and many bacteria. It provides the final electron sink by accepting electrons from cytochrome c and reducing oxygen to water (1). The energy generated from this reaction is used to translocate protons across the membrane against the membrane potential and pH gradient (2). The proton gradient so formed is then used to make ATP, a universal energy source. CcO is an intrinsic membrane protein with varying numbers of subunits from 3 in some bacteria to 13 in mammalian mitochondria. However, only subunits I and II, which contain the redox active heme and metal centers and are highly conserved among different species, are required for electron transfer and proton pumping activities. Other subunits, particularly the highly conserved subunit III, likely play key roles in stabilizing and regulating the enzyme activity and energy metabolism in general.Over the past decade, several x-ray crystal structures of aa 3 -type CcOs from bovine heart mitochondria and bacteria have been determined (3-8). However, the mechanism of vectorial translocation of protons by CcO remains to be solved (9). It is now recognized that water molecules play a critical role in facilitating and controlling proton transfer (10). Thus, highresolution crystal structures of different redox states and key mutants with defined waters are necessary to elucidate the energy conservation process. However, achieving a reproducible high-resolution structure of membrane proteins remains a ...

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Section: Resultsmentioning

confidence: 99%

Identification of conserved lipid/detergent-binding sites in a high-resolution structure of the membrane protein cytochrome c oxidase

Qin

Hiser

Mulichak

et al. 2006

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

Self Cite

284

385

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“…Histidine-tagged wild-type and mutant CcO were purified from R. sphaeroides, and the concentration was determined by using UV-visible spectroscopy as described (41). Further purification was done by using fast protein liquid chromatography (Amersham Pharmacia, AKTA-519) with tandem DEAE-5PW columns (Toso-Haas) (42).…”

Section: Methodsmentioning

confidence: 99%

Redox-coupled proton translocation in biological systems: Proton shuttling in cytochrome c oxidase

Namslauer

Pawate

Gennis

et al. 2003

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

113

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In the respiratory chain free energy is conserved by linking the chemical reduction of dioxygen to the electrogenic translocation of protons across a membrane. Cytochrome c oxidase (CcO) is one of the sites where this linkage occurs. Although intensively studied, the molecular mechanism of proton pumping by this enzyme remains unknown. Here, we present data from an investigation of a mutant CcO from Rhodobacter sphaeroides [Asn-139 3 Asp, ND(I-139)] in which proton pumping is completely uncoupled from the catalytic turnover (i.e., reduction of O 2). However, in this mutant CcO, the rate by which O 2 is reduced to H2O is even slightly higher than that of the wild-type CcO. The data indicate that the disabling of the proton pump is a result of a perturbation of E(I-286), which is located 20 Å from N(I-139) and is an internal proton donor to the catalytic site, located in the membranespanning part of CcO. The mutation results in raising the effective pK a of E(I-286) by 1.6 pH units. An explanation of how the mutation uncouples catalytic turnover from proton pumping is offered, which suggests a mechanism by which CcO pumps protons.

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“…Preparation of Cytochrome c Oxidase-Cytochrome c oxidase was purified from Rhodobacter sphaeroides as previously reported (15) using an Ni 2ϩ -NTA-agarose (Qiagen) affinity step followed by a further fast protein liquid chromatography (Amersham Biosciences, Inc., AKTA-519) purification procedure with tandem DEAE-5PW columns (Toso-Haas) (16). Bovine heart oxidase was purified by the Yoshikawa method (17 (22,23); and H93C/N I 2 and subunit III-less oxidase (24)) and purified in the same way as wild-type.…”

Section: Methodsmentioning

confidence: 99%

“…Alternatively, purified COVs were made using an oxidase construct with a His-tag on subunit II (htIICcO) that is able to bind to an Ni 2ϩ -NTA affinity chromatography column for isolation and concentration of COVs with correctly oriented oxidase, as described in Hiser et al (16). Basically, detergent was removed by a Sephadex G-25 column equilibrated with 75 mM HEPES-KOH, pH 7.4, ϩ14 mM KCl.…”

Section: Methodsmentioning

confidence: 99%

“…The electron donor was pre-reduced horse heart cytochrome c made by sodium dithionite reduction followed by desalting and depolymerizing through a Sephadex G-75 (Amersham Biosciences, Inc.) gel filtration 2 C. Hiser and S. Ferguson-Miller, unpublished. Proton Pumping Measurements-Proton pumping measurements were made as described previously (16). Scans were collected, and kinetic traces for cytochrome c oxidation at 550 nm or phenol red changes at 557 nm (isosbestic point of cytochrome c) were extracted after averaging at least three data sets and creating difference spectra (reduced minus oxidized).…”

Section: Methodsmentioning

confidence: 99%

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Membrane Potential-controlled Inhibition of Cytochromec Oxidase by Zinc

Mills

Schmidt

Hiser

et al. 2002

Journal of Biological Chemistry

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115

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Like many voltage-sensitive ion pumps, cytochrome c oxidase is inhibited by zinc. Binding of zinc to the outside surface of Rhodobacter sphaeroides cytochrome c oxidase inhibits the enzyme with a K I of < 5 M when the enzyme is reconstituted into phospholipid vesicles in the presence of a membrane potential. In the absence of a membrane potential and a pH gradient, millimolar concentrations of zinc are required to inhibit. This differential inhibition causes a dramatic increase in the respiratory control ratio from 6 to 40 for wild-type oxidase. 2؉ . Proton pumping is slower and less efficient with zinc. The results suggest that zinc inhibits proton movement through a proton exit path, which can allow proton back-leak at high membrane potentials. The physiological and mechanistic significance of proton movement in the exit pathway and its blockage by zinc is discussed in terms of regulation of the efficiency of energy transduction.

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C-Terminal Truncation and Histidine-Tagging of Cytochrome c Oxidase Subunit II Reveals the Native Processing Site, Shows Involvement of the C-Terminus in Cytochrome c Binding, and Improves the Assay for Proton Pumping

Cited by 38 publications

References 36 publications

Identification of conserved lipid/detergent-binding sites in a high-resolution structure of the membrane protein cytochrome c oxidase

Identification of conserved lipid/detergent-binding sites in a high-resolution structure of the membrane protein cytochrome c oxidase

Redox-coupled proton translocation in biological systems: Proton shuttling in cytochrome c oxidase

Membrane Potential-controlled Inhibition of Cytochromec Oxidase by Zinc

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