Conformations of cytochrome oxidase: thermodynamic evaluation of the interconversion of the 418- and 428-nm forms

Ja, Kornblatt; G, Hui Bon Hoa

doi:10.1021/bi00265a009

Cited by 18 publications

(4 citation statements)

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“…It was necessary to ensure that the pH did not change during the pressure experiments. Therefore, electrophoresis under pressure was performed using the Bistris buffer system, as the pKa of Bistris is almost independent of pressure [33,34]. We verified that the changes in the ultraviolet spectrum that occur upon addition of 6‐AH to plasminogen in phosphate buffer at pH 6.5–5 also occurred at pH 5 in Bistris.…”

Section: Methodsmentioning

confidence: 58%

The effects of hydrostatic pressure on the conformation of plasminogen

Kornblatt

Cléry³

et al. 1999

European Journal of Biochemistry

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Plasminogen undergoes a large conformational change when it binds 6-aminohexanoate. Using ultraviolet absorption spectroscopy and native PAGE, we show that hydrostatic pressure brings about the same conformational change. The volume change for this conformational change is 233 mL´mol 21 . Binding of ligand and hydrostatic pressure both cause the protein to open up to expose surfaces that had previously been buried in the interior.Keywords: conformational changes; hydrostatic pressure; plasminogen.Plasminogen, a glycoprotein present in vertebrate blood, carries out no known reactions. It is the inactive proenzyme form of plasmin, a protease of the chymotrypsin family. As a proenzyme, it is capable of binding to macromolecular or supramolecular surfaces which contain exposed carboxyterminal lysines or some exposed amino sugars. Having bound to a surface, the proenzyme undergoes a conformational change and can then be activated by any one of several proteases or other plasminogen molecules complexed to the same surface. Activation of the proenzyme involves cleavage of one peptide bond; the two chains remain joined by disulfide bonds [1]. The activated enzyme, plasmin, is one of the enzymes that cleaves fibrin. Native plasminogen is called gluplasminogen [2]. It is converted to several other plasminogen forms, termed lys-plasminogen, by cleavage of the amino terminus. The native structure contains < 790 amino acids [3]; it consists of an N-terminal domain, five kringles and a C-terminal proteolytic domain. The activation cleavage that gives rise to plasmin, occurs in the linker region between kringle 5 and the proteolytic domain. The secondary structure of plasminogen is interesting: it includes the five kringles, all of which are quite similar; each contains < 80 amino acids packed as four pairs of antiparallel b-sheets with b-linkers as shown by far UV CD [4], and confirmed by both X-ray crystallography [5,6] and solution NMR of isolated kringles [7]. The name for this folding pattern derives from the Danish word for pretzel. The protease domain has the same tertiary structure as that of the pancreatic serine proteases [8]. Conformational changes occur when plasminogen binds C-terminal lysine derivatives or analogous compounds, such as 6-aminohexanoate (6-AH). It is believed that the binding of these small molecules mimics the physiological binding of macromolecular surfaces such as that of the fibrin clot. At least three 6-AH can bind. Binding to tightest site (kringle 1) does not cause a detectable conformational change [9] but binding to kringles 4 and 5 results in major structural changes [10] for glu-plasminogen and minor changes for the lys-plasminogens. Small angle neutron scattering indicated that when glu-plasminogen binds 6-AH the protein changes from a prolate ellipsoid with a radius of gyration of 39 A Ê and maximum dimension of 147 A Ê to one with radius of gyration of 56 A Ê and maximum dimension of < 250 A Ê [4]. Low angle X-ray scattering showed a more compact structure for the unligated form [11] ...

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Section: Methodsmentioning

confidence: 58%

The effects of hydrostatic pressure on the conformation of plasminogen

Kornblatt

Cléry³

et al. 1999

European Journal of Biochemistry

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“…Properties of Cytochrome c Oxidase. The behavior of the oxidase under pressure has been described (Kornblatt & Hui Bon Hoa, 1982). The protein undergoes a reversible highspin/low-spin transition when subjected to pressure, the °of which varies between 4 and 8 mL mol-1 depending on the temperature.…”

Section: Resultsmentioning

confidence: 99%

Volume changes associated with cytochrome c oxidase-porphyrin cytochrome c equilibrium

Kornblatt¹,

Hoa²,

English³

1984

Biochemistry

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The binding of a fluorescent derivative of cytochrome c to cytochrome c oxidase has been studied by use of pressure to perturb the equilibrium. delta Vo for the reaction oxidase-porphyrin cytochrome (formula; see text) was small and favored dissociation of the complex. Pressure-induced dissociation is to be expected if the major forces governing the equilibrium are electrostatic in nature. The dependence of log Kd on pressure is not linear but biphasic; high pressures lead to a decrease in Kd and association of the reactants. The latter fact indicates that the net compressibility of the complexes is greater than that of the free reactants, an unexpected result.

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“…In this work, elevated hydrostatic pressure was utilized to probe for differential structural and functional stability between monomeric and dimeric CcO. This technique had been used previously only to probe for volume changes associated with conformational switching within detergent-solubilized CcO and to assess the functional role of bound water within the enzyme ( − ). No attention has been paid to determining the effect of exposure to elevated hydrostatic pressure on either the quaternary protein structure or the electron transport activity.…”

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

et al. 2007

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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...

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Conformations of cytochrome oxidase: thermodynamic evaluation of the interconversion of the 418- and 428-nm forms

Cited by 18 publications

References 40 publications

The effects of hydrostatic pressure on the conformation of plasminogen

The effects of hydrostatic pressure on the conformation of plasminogen

Volume changes associated with cytochrome c oxidase-porphyrin cytochrome c equilibrium

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

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