Uncouplers protonophoric

Carbonylcyanide-4-trilluoromethoxyphenylhydrazone is known as a protonophore or uncoupler of oxidative phosphorylation in bioelectrochemistry because it disrupts the tight coupling between electron transport and the ATP synthase. Uncouplers act by dis-... 

A relationship correlating the weak acid uncouplers activity with their A %-, pAi°, and has been presented on the basis of protonophoric theory of uncoupling activity, in which the concentration of anionic ionophore (A ) within a biomembrane is supposed to be controlled by the ionic partition of A at the biomembrane solution interface according to Eq. (28) [19]. The biomembrane solution interface could be polarized or electrogenic [37]. Experimental results on the activities of uncouplers on rat liver mitochondria [30] have been explained reasonably [19,24]. 

Miyoshi, H., Nishioka, T. and Fujita, T. (1987). Quantitative relationship between protonophoric and uncoupling activities of substituted phenols, Biochim. Biophys. Acta, 891, 194-204. 

Uncoupling in hamster brown adipocytes by a natural protonophore does not appear to cause simultaneous aerobic and anaerobic processes because Nedergaard et al. (1977) reported that these cells stimulated by norepinephrine (see p. 320) had... 

B. Beauvoit, M. Rigoulet, G. Raffard, P. Canioni, and B.Guerin (1991). Differential sensitivity of the cellular compartments of Saccharomyces cerevisiae to protonophoric uncoupler under fermentative and respiratory energy supply. Biochemistry, 30, 11212-11220. 

Each thus interacts with the other, squiggle s just p.m.f. and no more. An uncoupler simply functions by acting as protonophore. 

The above reasoning was recently confirmed by several pieces of indirect evidence. It was found that in H. halobium (i) protonophorous uncouplers decrease A/in and cause a repellent effect [45], (ii) cyanide and DCCD have no effect on the photoresponse of a mutant which possesses sensory rhodopsins but no bacteriorhodopsin [48], and (iii) in a similar mutant, A l is not involved in photosensing [49]. 

Perhaps it is the Na cycle that is responsible for the effect observed by Michel and Oesterhelt[109] (see also [110]) who reported that the protonophorous uncoupler abolishes A/Ih+ at much lower concentrations than those affecting the phosphate potential. 

The possibility that primary Na extrusion was driven by a Na -translocating ATPase was excluded The protonophore tetrachlorosalicylanilide was found to uncouple formaldehyde oxidation from ATP synthesis without affecting Na extrusion. Thus, ATP cannot be the driving force for Na extrusion in Methanosarcina barken. This situation may be different in Methanococcus voltae which appears to contain a Na -translocating ATPase (see above). 

Depending on whether the source of energy is the respiratory chain or added ATP, the uptake of Ca " is inhibited by respiratory chain inhibitors or oligomycin, respectively. As expected, protonophoric uncouplers inhibit in both cases. The competitive inhibition by Mg [29], is of particular interest, since it may have physiological significance due to the presenee of Mg " in the cytosol of cells. Mg, however, is not transported by mitochondria through the Ca " -uptake system which, on the other hand, translocates Mn, Ba and Sr (see Refs. 13-15 for reviews). 

Uncoupling results in dissipation of the respiratory chain-produced ApH+ due to increased conductance of the inner mitochondrial membrane. Thus energy released by respiration is immediately dissipated as heat without formation and hydrolysis of ATP. Non-esterified fatty acids proved to be compounds mediating the thermoregulatory uncoupling. They operate as protonophorous uncouplers with the help of special uncoupling proteins (UCPs) or some mitochondrial antiporters i.e. the ATP/ADP antiporter and aspartate/glutamate antiporter [1-5]. 

The process proved to be inhibited by even a small ApH+ decrease ( mild uncoupling ) [5]. It was suggested that mild uncoupling is carried out by free fatty acids operating as protonophores with the help of UCPs and ATP/ADP-antiporter [5]. 

Uncoupling of oxidative phosphorylation by 2,4-dinitrophenol (2,4-DNP). The anionic form of 2,4-DNP is protonated in the intermembrane space, is lipid soluble, and crosses the inner membrane readily. In the matrix, the protonated form dissociates, abolishing the proton gradient established by substrate oxidation. The ionized form of 2,4-DNP is poorly soluble in the membrane lipids and therefore is not easily transported across the membrane (dashed arrow). It is lipophilic and capable of transporting protons from one side of the membrane to the other (a protonophore), thus abolishing the proton gradient. 

Highly lipophilic weak acids and bases that have the capacity to remain lipophilic in both their protonated and deprotonated forms can act as protonophores. Such compounds belong to another class of ionophores that are often referred to as mitochondrial uncouplers because of their unique ability to translocate protons across mitochondrial membranes, resulting in the subsequent loss of the mitochondrial proton gradient that is required to drive oxidative phosphorylation. While certain natural products act as mitochondrial uncouplers, most of the protonophores used as pharmacological probes are not natural products but are low-molecular-weight synthetic compounds (e.g., carbonyl cyanide -trifluoromethoxyphenylhydrazone (FCCP)). 

Fromenty B, Fisch C, Berson A, Letteron P, Larrey D, Pessayre D (1990b) Dual effect of amiodarone on mitochondrial respiration. Initial protonophoric uncoupling effect followed by inhibition of the respiratory chain at the levels of complex I and complex II. J Pharmacol Exp Ther 255 1377-1384... 

NO3 > Br > Cl > I acetate isethionate. The same is true for the valinomycin-stimulated ATPase reaction. The vesicular gradient for was abohshed by a combination of a K -ionophore and a protonophore or a K - H exchange ionophore (nigericin). Under these circumstances the ATPase is uncoupled and does not generate proton transport. 

Many different protonophores, each with a different effectiveness, are available like 2,4-dinitrophenol (DNP) or 5-chloro-3-tert-butyl-2 -chloro-4 -nitro-salicyl-anilide (CCCP). Manipulation of the two components of the A/Ih can be effected with two potassium ionophores. Valinomycin increases the electrogenic permeability of a membrane for potassium and leads to the dissipation of the if a high concentration of potassium is present (more than 10 mM). Nigericin catalyzes an electroneutral potassium proton exchange and thus dissipates the A pH under the same conditions. In combination these two ionophores function as an uncoupler. 

Long-chain fatty acids also cause uncoupling. The mechanism appears complex, but seems to be distinct from either simple membrane disruption or a classical protonophoric effect, and appears to require the involvement of active transporters [9, 14, 19, 21-24). 

Although much less commonly observed, there have been several reports of lipophilic weak bases acting as protonophoric uncouplers, e.g., the pyridine AU-1421 (1) [30]. These compounds can transport protons as shown in Fig. 13.4.2. The weakly basic compound is protonated on the low pH intermembrane space side of the membrane, and the resulting cation (BH+) crosses the membrane, driven by the transmembrane potential. In the less acidic matrix, the proton is removed, and the neutral uncoupler (B) can diffuse back across the membrane to continue the cyde. 

Although little resistance to uncouplers has been observed in fungi, several bacteria are known that have reduced sensitivity to protonophoric uncouplers [78-80]. The likelihood of similar resistance mechanisms developing in fungi is unknown, but is probably small, as no such mutations have yet been observed. 

To act as an efficient protonophoric uncoupler a weakly acidic compound must have properties that allow it, in both the uncharged protonated and the anionic deprotonated forms, to enter and cross the membrane lipid bilayer. The compound must have a suitable plQ such that on the more acidic, intermembrane side of the membrane a significant proportion is protonated, whilst on the less acidic matrix side a proportion is deprotonated. A compound that is not acidic enough may transfer a single proton across the membrane, but will not release it in the matrix, and hence cannot repeat the cyde. In contrast, too strong an acid will remain deprotonated even in the intermembrane space. For this reason 2,4-dinitrophenol (pJCa 4.04) is a stronger uncoupler than 2-nitrophenol (plQ 7.14) and picric acid (pKa 0.53) does not act as a protonophoric uncoupler in mitochondria, although all three compounds have a similar lipophilidty [88]. 

To efficiently cross the membrane, uncouplers must be reasonably lipophilic (generally log P > 2). However, this alone is not suffident. A key part of the action of a protonophoric uncoupler is the ability to cross the membrane in the anionic form, and this requires the negative charge to be extensively delocalized, or shielded from the lipid interior of the membrane in some way. For this reason, many of the most potent uncouplers combine bulky lipophilic groups adjacent to the ionizable proton with extended conjugated systems through which the charge can be spread. A typical example of this is malonoben (10), one of the most potent phenolic uncouplers known (Fig. 13.4.3) [100]. 

However, many lipophilic carboxylic acids satisfy the log P and pKa criteria for uncoupling, but the charge on the anion cannot be delocalized beyond the carboxyl group and so they do not act as effective protonophoric uncouplers. Those that do have structural features that allow the charge to be delocalized include the anacardic acids, where the adjacent phenol stabilizes the negative charge (Fig. 13.4.4a) [101]. A similar intramolecular interaction occurs in the salicylanilide class of uncouplers, of which one of the most active is S-13 (11, Fig. 13.4.4b). 

Hydrazones prepared by the reaction of aryldiazonium salts with malononitrile (trivially named carbonyl cyanide phenylhydrazones, or CCPs) have been known as uncouplers for many years [114]. Studies on the relationship between physicochemical properties and uncoupling potency have been reported [90, 91], and these show that this relationship is very similar to those for other classes of weakly acidic protonophoric uncouplers. Thus, two of the most potent uncouplers of... 

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