Rapid purification of wildtype and mutant cytochrome c oxidase from Rhodobacter sphaeroides by Ni2+‐NTA affinity chromatography

Mitchell, David M.; Gennis, Robert B.

doi:10.1016/0014-5793(95)00626-k

Cited by 156 publications

(131 citation statements)

References 29 publications

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“…CcO with a 6× His-tag fused to the carboxy-terminus of subunit I was homologously expressed in R. sphaeroides and purified according to ref 38. CcO adsorbs to the Ni-NTA moiety via the coordination of the nitrogen of two of the imidazole side chains of the 6× His-tag ( Figure 1C).…”

Section: Methodsmentioning

confidence: 99%

Oriented Attachment and Membrane Reconstitution of His-Tagged CytochromecOxidase to a Gold Electrode: In Situ Monitoring by Surface-Enhanced Infrared Absorption Spectroscopy

et al. 2004

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Abstract:A novel concept is introduced for the oriented incorporation of membrane proteins into solid supported lipid bilayers. Recombinant cytochrome c oxidase solubilized in detergent was immobilized on a chemically modified gold surface via the affinity of its histidine-tag to a nickel-chelating nitrilo-triacetic acid (NTA) surface. The oriented protein monolayer was reconstituted into the lipid environment by detergent substitution. The individual steps of the surface modification, including (1) chemical modification of the gold support, (2) adsorption of the protein, and (3) reconstitution of the lipid bilayer, were followed in situ by means of surface-enhanced infrared absorption spectroscopy (SEIRAS) and accompanied by normalmode analysis. The high surface sensitivity of SEIRAS allows for the identification of each chemical reaction process within the monolayer at the molecular level. Finally, full functionality of the surface-tethered cytochrome c oxidase was demonstrated by cyclic voltammetry after binding of the natural electron donor cytochrome c.

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Section: Methodsmentioning

confidence: 99%

Oriented Attachment and Membrane Reconstitution of His-Tagged CytochromecOxidase to a Gold Electrode: In Situ Monitoring by Surface-Enhanced Infrared Absorption Spectroscopy

et al. 2004

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“…Site-directed mutagenesis was performed as described (53). R. sphaeroides was grown in Sistrom medium and the His-tagged protein was purified on a Ni column as described (54). After elution, the buffer was exchanged to 0.1 M Hepes pH 7.4, 0.1% dodecyl β-d-maltoside (DDM) before freezing in liquid nitrogen.…”

Section: Methodsmentioning

confidence: 99%

Functional interactions between membrane-bound transporters and membranes

Öjemyr

Lee

Gennis

et al. 2010

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

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One key role of many cellular membranes is to hold a transmembrane electrochemical ion gradient that stores free energy, which is used, for example, to generate ATP or to drive transmembrane transport processes. In mitochondria and many bacteria, the gradient is maintained by proton-transport proteins that are part of the respiratory (electron-transport) chain. Even though our understanding of the structure and function of these proteins has increased significantly, very little is known about the specific role of functional protein-membrane and membrane-mediated proteinprotein interactions. Here, we have investigated the effect of membrane incorporation on proton-transfer reactions within the membrane-bound proton pump cytochrome c oxidase. The results show that the membrane acts to accelerate proton transfer into the enzyme's catalytic site and indicate that the intramolecular proton pathway is wired via specific amino acid residues to the twodimensional space defined by the membrane surface. We conclude that the membrane not only acts as a passive barrier insulating the interior of the cell from the exterior solution, but also as a component of the energy-conversion machinery.cytochrome aa3 | electron transfer | energy transduction | membrane protein | respiration

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“…The N139D mutant is missing the "intermediate" electrogenic phase which thus can be tentatively identified as vectorial transfer of the "pumped" proton within the protein in the direction of the outer aqueous phase across part of the insulating barrier. Wild type and mutant cytochrome c oxidase, modified by a sixhistidine affinity tag, was purified from R. sphaeroides by chromatography on a nickel column as described previously (16). Purified cytochrome c oxidase was reconstituted in phospholipid (asolectin) vesicles by a cholate dialysis method (17).…”

mentioning

confidence: 99%

Transmembrane Charge Separation during the Ferryl-oxo → Oxidized Transition in a Nonpumping Mutant of Cytochrome c Oxidase

Siletsky

Pawate

Weiss³

et al. 2004

Journal of Biological Chemistry

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Cytochrome c oxidase (COX) 1 is the terminal enzyme of the aerobic respiratory chain in mitochondria and in many prokaryotes (1-4). The enzyme conducts electrons from cytochrome c to O 2 and couples the energetically downhill electron transfer to the uphill electrogenic movement of protons across the membrane from the bacterial cytoplasm (or mitochondrial matrix) to the periplasm (or mitochondrial intermembrane space).The transmembrane charge separation by COX involves two different mechanisms. Half of the free energy (corresponding to the transmembrane translocation of four elementary electric charges per dioxygen reduced) is conserved by a direct mechanism; the four electrons and four protons required to convert O 2 to water come from opposite sides of the membrane. This direct mechanism was postulated by Mitchell (5, 6) in the original chemiosmotic hypothesis. Charge separation via this mechanism is an intrinsic part of the reaction chemistry and cannot be dissociated from it without re-engineering the proton and/or electron delivery pathways to the active site. Any mutation resulting in inhibition of this electrogenic function would inevitably inhibit electron transfer activity of the enzyme.The other half of the free energy is conserved by virtue of "proton pumping" discovered by Wikström in 1977 (7) and resulting in the transmembrane electrogenic translocation of four more protons per catalytic cycle. This proton pumping occurs by an "indirect" coupling mechanism and is not an obligatory part of the reaction chemistry. In principle, residues constituting the proton pump may be located in a separate part of the protein from the catalytic center, and the coupling can occur by virtue of finely tuned mechano-electrical interactions mediated by minor conformational changes of the protein (cf. Refs. 8 and 9).The indirect coupling of the proton pump has been demonstrated recently by the isolation of cytochrome oxidase mutants that have full catalytic electron transfer activity but that do not pump protons. This was first shown for the N131D mutant of the COX from Paracoccus denitrificans (10), which places an aspartic acid residue near the entrance of the D-channel. This mutant COX has wild type oxidase activity but does not pump protons (10), which is exactly the phenotype expected for cytochrome oxidase in which electron transfer is decoupled from proton translocation at the molecular level. The equivalent mutant of the COX from Rhodobacter sphaeroides, N139D,

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Rapid purification of wildtype and mutant cytochrome c oxidase from Rhodobacter sphaeroides by Ni²⁺‐NTA affinity chromatography

Cited by 156 publications

References 29 publications

Oriented Attachment and Membrane Reconstitution of His-Tagged CytochromecOxidase to a Gold Electrode: In Situ Monitoring by Surface-Enhanced Infrared Absorption Spectroscopy

Oriented Attachment and Membrane Reconstitution of His-Tagged CytochromecOxidase to a Gold Electrode: In Situ Monitoring by Surface-Enhanced Infrared Absorption Spectroscopy

Functional interactions between membrane-bound transporters and membranes

Transmembrane Charge Separation during the Ferryl-oxo → Oxidized Transition in a Nonpumping Mutant of Cytochrome c Oxidase

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