Exposure of Bovine Cytochrome c Oxidase to High Triton X-100 or to Alkaline Conditions Causes a Dramatic Change in the Rate of Reduction of Compound F

Sadoski, Robert C.; Zaslavsky, Dmitry; Gennis, Robert B.; Durham, Bill; Millett, Francis

doi:10.1074/jbc.m103640200

Cited by 23 publications

(16 citation statements)

References 26 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This order of reactions is the reverse compared with that observed in the present study with R. sphaeroides CcO. However, it should be noted that in monomeric mitochondrial CcO the pH titration of the F 3 3 O 4 transition rate is shifted to higher pH values as compared with the dimeric form of the enzyme (49). This result indicates that around pH 7 proton uptake is not rate-limiting for the F 3 3 O 4 transition and that there is an internal proton pool in the monomeric enzyme (31).…”

Section: Discussioncontrasting

confidence: 44%

The timing of proton migration in membrane-reconstituted cytochrome c oxidase

Salomonsson

Faxén

Ädelroth

et al. 2005

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

View full text Add to dashboard Cite

In mitochondria and aerobic bacteria energy conservation involves electron transfer through a number of membrane-bound protein complexes to O2. The reduction of O2, accompanied by the uptake of substrate protons to form H2O, is catalyzed by cytochrome c oxidase (CcO). This reaction is coupled to proton translocation (pumping) across the membrane such that each electron transfer to the catalytic site is linked to the uptake of two protons from one side and the release of one proton to the other side of the membrane. To address the mechanism of vectorial proton translocation, in this study we have investigated the solvent deuterium isotope effect of proton-transfer rates in CcO oriented in small unilamellar vesicles. Although in H2O the uptake and release reactions occur with the same rates, in D2O the substrate and pumped protons are taken up first (D Х 200 s, ''peroxy'' to ''ferryl'' transition) followed by a significantly slower proton release to the other side of the membrane (D Х 1 ms). Thus, the results define the order and timing of the proton transfers during a pumping cycle. Furthermore, the results indicate that during CcO turnover internal electron transfer to the catalytic site is controlled by the release of the pumped proton, which suggests a mechanism by which CcO orchestrates a tight coupling between electron transfer and proton translocation.heme-copper oxidases ͉ electron transfer ͉ membrane protein ͉ respiratory chain I n membrane-bound proton pumps the endergonic proton translocation across the membrane is driven by an exergonic reaction, e.g., electron transfer from a low-potential donor to a high-potential acceptor. The protons are translocated through proton-transfer pathways, typically composed of networks of hydrogen-bonded protonatable and polar amino acid side chains and bound water molecules, that span the transmembrane part of the protein. To prevent short-circuiting of the electrochemical gradient it is crucial that there is never simultaneous access (i.e., a direct contact) to the two membrane sides. One way to control the proton access is to alter the position of e.g., an amino acid side chain such that the group exchanges protons rapidly with either one side or the other side of the membrane (but never both sides simultaneously), defining an input and an output state (for review, see refs. 1-6). Proton pumping would be accomplished, for example, if the reaction that drives proton translocation is energetically coupled to changes in the pK a value of the protonatable group such that it has a high pK a in the input state and a low pK a in the output state. In the discussion below, we will refer to such a group as a ''pumping element.'' Cytochrome c oxidase (CcO), which belongs to the hemecopper oxidase superfamily, is an example of a redox-driven proton pump (for review, see refs 4, 5, and 7-12). The CcOs are found in the cytoplasmic membrane in bacteria or the inner membrane of mitochondria, where they catalyze the reduction of molecular oxygen to water. The electrons are donated by...

show abstract

Section: Discussioncontrasting

confidence: 44%

The timing of proton migration in membrane-reconstituted cytochrome c oxidase

Salomonsson

Faxén

Ädelroth

et al. 2005

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

View full text Add to dashboard Cite

show abstract

“…The yield of the F state was determined spectroscopically (as described in ref. 22) to be 65-80% in different preparations. The laser kinetic experiments were carried out within 5 min of treatment with H 2 O 2 , and there was no loss of steady-state electron transfer activity because of modification of the enzyme within this time period (25).…”

Section: Methodsmentioning

confidence: 99%

“…Briefly exposing the enzyme to either 5% Triton X-100 at pH 8 or alkaline pH (pH 10) converts the normal dimeric form of the bovine enzyme into a monomeric species that has full steady-state and proton-pumping activities and renders the F3O transition pH-independent up to at least pH 10.5 (22). The unusual kinetic properties of the monomeric bovine oxidase result from the selective inhibition of proton uptake from solution.…”

mentioning

confidence: 99%

Direct measurement of proton release by cytochrome c oxidase in solution during the F→O transition

Zaslavsky

Sadoski

Rajagukguk

et al. 2004

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

Self Cite

View full text Add to dashboard Cite

The mechanism by which electron transfer is coupled to proton pumping in cytochrome c oxidase is a major unsolved problem in molecular bioenergetics. In this work it is shown that, at least under some conditions, proton release from the enzyme occurs before proton uptake upon electron transfer to the heme͞Cu active site of the enzyme. This sequence is similar to that of proton release and uptake observed for the light-activated proton pump bacteriorhodopsin. In the case of cytochrome c oxidase, this observation means that both the ejected proton and the proton required for the chemistry at the enzyme active site must come from an internal proton pool.

show abstract

“…Monomeric CcO dimerizes during its reconstitution into phospholipid vesicles, precluding assessment of proton translocation with monomeric CcO (6,7). However, dimerization slows the reduction of catalytic intermediate compound F, suggesting that uptake of protons into the binuclear center is slower with dimeric CcO (8).…”

mentioning

confidence: 99%

Differential Stability of Dimeric and Monomeric Cytochrome c Oxidase Exposed to Elevated Hydrostatic Pressure

et al. 2007

View full text Add to dashboard Cite

Detergent-solubilized dimeric and monomeric cytochrome c oxidase (CcO) have significantly different quaternary stability when exposed to 2−3 kbar of hydrostatic pressure. Dimeric, dodecyl maltoside-solubilized cytochrome c oxidase is very resistant to elevated hydrostatic pressure with almost no perturbation of its quaternary structure or functional activity after release of pressure. In contrast to the stability of dimeric CcO, 3 kbar of hydrostatic pressure triggers multiple structural and functional alterations within monomeric cytochrome c oxidase. The perturbations are either irreversible or slowly reversible since they persist after the release of high pressure. Therefore, standard biochemical analytical procedures could be used to quantify the pressure-induced changes after the release of hydrostatic pressure. The electron transport activity of monomeric cytochrome c oxidase decreases by as much as 60% after exposure to 3 kbar of hydrostatic pressure. The irreversible loss of activity occurs in a time-and pressure-dependent manner. Coincident with the activity loss is a sequential dissociation of four subunits as detected by sedimentation velocity, highperformance ion-exchange chromatography, and reversed-phase and SDS-PAGE subunit analysis. Subunits VIa and VIb are the first to dissociate followed by subunits III and VIIa. Removal of subunits VIa and VIb prior to pressurization makes the resulting 11-subunit form of CcO even more sensitive to elevated hydrostatic pressure than monomeric CcO containing all 13 subunits. However, dimeric CcO, in which the association of VIa and VIb is stabilized, is not susceptible to pressure-induced inactivation. We conclude that dissociation of subunit III and/or VIIa must be responsible for pressure-induced inactivation of CcO since VIa and VIb can be removed from monomeric CcO without significant activity loss. These results are the first to clearly demonstrate an important structural role for the dimeric form of cytochrome c oxidase, i.e., stabilization of its quaternary structure.Bovine heart cytochrome c oxidase (EC 1.9.3.1, CcO) 1 is the terminal complex of the mitochondrial respiratory chain. It is a multisubunit protein-phospholipid complex consisting of 13 dissimilar subunits, three or four tightly bound cardiolipins, and four metal centers (Cu A , heme a, heme a 3 , and Cu B ) with a combined molecular weight of ∼205000 for the monomeric enzyme (1-3). CcO crystallizes as a dimer, which is generally seen as the functional unit within the inner membrane; however, little or no information is available regarding the functional or structural significance of a dimeric structure. For example, monomeric and dimeric CcO are both highly active in terms of electron transfer (4,5). The dimeric form of † This work was supported by grants from the National Institutes of Health (GMS 24795) and The Robert A. Welch Foundation (AQ1481).* To whom correspondence should be addressed. Telephone: (210) 567−3754. Fax: (210) 567−6595. E-mail: robinson@uthscsa.edu. ‡ Present add...

show abstract

Exposure of Bovine Cytochrome c Oxidase to High Triton X-100 or to Alkaline Conditions Causes a Dramatic Change in the Rate of Reduction of Compound F

Cited by 23 publications

References 26 publications

The timing of proton migration in membrane-reconstituted cytochrome c oxidase

The timing of proton migration in membrane-reconstituted cytochrome c oxidase

Direct measurement of proton release by cytochrome c oxidase in solution during the F→O transition

Differential Stability of Dimeric and Monomeric Cytochrome c Oxidase Exposed to Elevated Hydrostatic Pressure

Contact Info

Product

Resources

About