On the mechanism of action of oligomycin and acidic uncouplers on proton translocation and energy transfer in “sonic” submitochondrial particles

A study is presented of the effect of acidic uncouplers and oligomycin on energy-linked and passive proton translocation, oxidative phosphorylation, and energy-linked nicotinamide-adenine-nucleotide transhydrogenase in EDTA submitochondrial particles from beef-heart. A flow potentiometric technique has been applied to resolve the kinetics of the initial rapid phase of the redox proton pump. Rapid kinetics analysis shows that carbonyl-cyanide-p-trifluoromethoxyphenyl-hydrazone (FCCP) does not exert any direct effect on redox-linked active proton transport. The uncoupling action of FCCP on oxidative phosphorylation and energy-linked transhydrogenase is shown to be quantitatively accounted for by its promoting effect of passive proton-diffusion across the mitochondrial membrane. Oligomycin depresses passive proton diffusion in EDTA sonic particles and this effect accounts for the coupling action exerted by the antibiotic on oxidative phosphorylation and energy-linked transhydrogenase. In fact, rapid kinetic analysis demonstrates that oligomycin does not directly affect the redox-linked proton pump. The present results show that there does not exist any labile intermediate in the redox-linked proton pump which is sensitive to acidic uncouplers.     

The proton-pumping site of cytochrome c oxidase: a model of its structure and mechanism

Cytochrome c oxidase is an electron-transfer driven proton pump. In this paper, we propose a complete chemical mechanism for the enzyme's proton-pumping site. The mechanism achieves pumping with chemical reaction steps localized at a redox center within the enzyme; no indirect coupling through protein conformational changes is required. The proposed mechanism is based on a novel redox-linked transition metal ligand substitution reaction. The use of this reaction leads in a straightforward manner to explicit mechanisms for achieving all of the processes previously determined (Blair, D.F., Gelles, J. and Chan, S.I. (1986) Biophys. J. 50, 713-733) to be needed to accomplish redox-linked proton pumping. These processes include: (1) modulation of the energetics of protonation/deprotonation reactions and modulation of the energetics of redox reactions by the structural state of the pumping site; (2) control of the rates of the pump's redox reactions with its electron-transfer partners during the turnover cycle (gating of electrons); and (3) regulation of the rates of the protonation/deprotonation reactions between the pumping site and the aqueous phases on the two sides of the membrane during the reaction cycle (gating of protons). The model is the first proposed for the cytochrome oxidase proton pump which is mechanistically complete and sufficiently specific that a realistic assessment can be made of how well the model pump would function as a redox-linked free-energy transducer. This assessment is accomplished via analyses of the thermodynamic properties and steady-state kinetics expected of the model. These analyses demonstrate that the model would function as an efficient pump and that its behavior would be very similar to that observed of cytochrome oxidase both in the mitochondrion and in purified preparations. The analysis presented here leads to the following important general conclusions regarding the mechanistic features of the oxidase proton pump. (1) A workable proton-pump mechanism does not require large protein conformational changes. (2) A redox-linked proton pump need not display a pH-dependent midpoint potential, as has frequently been assumed. (3) Mechanisms for redox-linked proton pumps that involve transition metal ligand exchange reactions are quite attractive because such reactions readily lend themselves to the linked gating processes necessary for proton pumping.     

Protonmotive force generation by a redox loop mechanism

Respiration involves the oxidation and reduction of substrate for the redox-linked formation of a protonmotive force (PMF) across the inner membrane of mitochondria or the plasma membrane of bacteria. A mechanism for PMF generation was first suggested by Mitchell in his chemiosmotic theory. In the original formulations of the theory, Mitchell envisaged that proton translocation was driven by a 'redox loop' between two catalytically distinct enzyme complexes. Experimental data have shown that this redox loop does not operate in mitochondria, but has been confirmed as an important mechanism in bacteria. The nitrate respiratory pathway in Escherichia coli is a paradigm for a protonmotive redox loop. The structure of one of the enzymes in this two-component system, formate dehydrogenase-N, has revealed the structural basis for the PMF generation by the redox loop mechanism and this forms the basis of this review.     

Prevention of leak in the proton pump of cytochrome c oxidase

The cytochrome c oxidases (CcO), which are responsible for most O(2) consumption in biology, are also redox-linked proton pumps that effectively convert the free energy of O(2) reduction to an electrochemical proton gradient across mitochondrial and bacterial membranes. Recently, time-resolved measurements have elucidated the sequence of events in proton translocation, and shed light on the underlying molecular mechanisms. One crucial property of the proton pump mechanism has received less attention, viz. how proton leaks are avoided. Here, we will analyse this topic and demonstrate how the key proton-carrying residue Glu-242 (numbering according to the sequence of subunit I of bovine heart CcO) functions as a valve that has the effect of minimising back-leakage of the pumped proton.     

A new redox loop formality involving metal-catalysed hydroxide-ion translocation. A hypothetical Cu loop mechanism for cytochrome oxidase

A new hypothetical type of redox loop is described, which translocates hydroxide instead of protons. Conventional protonmotive redox loops use carriers of protons with electrons (e.g. QH2/Q systems) to couple electron transfer to the translocation of protons. The putative hydroxidemotive redox loop uses carriers of hydroxide ions against electrons (e.g. transition-metal centres) to couple electron transfer to the translocation of hydroxide ions. This simple idea leads to the proposal of a hydroxidemotive Cu loop mechanism that may possibly be applicable to the CuA or CuB centre of cytochrome oxidase, and might thus account for the coupling of electron transfer to net proton translocation in that osmoenzyme.     

Discrete steps in dioxygen activation—The cytochrome oxidase/O2 reaction

The kinetic constraints that are imposed on cytochrome oxidase in its dual function as the terminal oxidant in the respiratory process and as a redox-linked proton pump provide a unique opportunity to investigate the molecular details of biological O₂ activation. By using flow/flash techniques, it is possible to visualize individual steps in the O₂-binding and reduction process, and results from a number of spectroscopic investigations on the oxidation of reduced cytochrome oxidase by O₂ are now available. In this article, we use these results to synthesize a reaction mechanism for O₂ activation in the enzyme and to simulate time-concentration profiles for a number of intemediates that have been observed experimentally. Kinetic manifestation of the consequences of coupling exergonic electron transfer to endergonic proton translocation emerge from this analysis. Energetic efficiency in this process apparently requires that potentially toxic intermediate oxidation states of dioxygen accumulate to substantial concentration during the reduction reaction.     

Respiratory Complex I: Structure, Redox Components, and Possible Mechanisms of Energy Transduction

Structural arrangements and properties of redox components of the mitochondrial and bacterial proton-translocating NADH:quinone oxidoreductases are briefly described. A model for the mechanism of proton translocation at first coupling site, which emphasizes participation of specifically Complex I-associated ubisemiquinones, is discussed. An alternative mechanism is proposed where all redox reactions take place in a hydrophilic part of the enzyme and the free energy accumulated as conformational constraint drives the proton pump associated with the hydrophobic polypeptides.     

Cytochrome oxidase as a proton pump

The general structure of cytochrome oxidase is reviewed and evidence that the enzyme acts as a redox-linked proton pump outlined. The overall H⁺/e ^– stoichiometry of the pump is discussed and results [Wikström (1989),Nature 338, 293] which suggest that only the final two electrons which reduce the peroxide adduct to water are coupled to protein translocated are considered in terms of the restrictions they place on pump mechanisms. Direct and indirect mechanisms for proton translocation are discussed in the context of evidence for redox-linked conformational changes in the enzyme, the role of subunit III, and the nature of the Cu_A site.     

Proton translocation by cytochrome c oxidase in different phases of the catalytic cycle

Since mitochondrial cytochrome c oxidase was found to be a redox-linked proton pump, most enzymes of the haem-copper oxidase family have been shown to share this function. Here, the most recent knowledge of how the individual reactions of the enzyme's catalytic cycle are coupled to proton translocation is reviewed. Two protons each are pumped during the oxidative and reductive halves of the cycle, respectively. An apparent controversy that concerns proton translocation during the reductive half is resolved. If the oxidised enzyme is allowed to relax in the absence of reductant, the binuclear haem-copper centre attains a state that lies outside the main catalytic cycle. Reduction of this form of the enzyme is not linked to proton translocation, but is necessary for a return to the main cycle. This phenomenon might be related to the previously described "pulsed" vs. "resting" and "fast" vs."slow" forms of haem-copper oxidases.     

The organisation of proton motive and non-proton motive redox loops in prokaryotic respiratory systems

Respiration is fundamental to the aerobic and anaerobic energy metabolism of many prokaryotic and most eukaryotic organisms. In principle, the free energy of a redox reaction catalysed by a membrane-bound electron transport chain is transduced via the generation of an electrochemical ion (usually proton) gradient across a coupling membrane that drives ATP synthesis. The proton motive force (pmf) can be built up by different mechanisms like proton pumping, quinone/quinol cycling or by a redox loop. The latter couples electron transport to a net proton transfer across the membrane without proton pumping. Instead, charge separation is achieved by quinone-reactive enzymes or enzyme complexes whose active sites for substrates and quinones are situated on different sides of the coupling membrane. The necessary transmembrane electron transport is usually accomplished by the presence of two haem groups that face opposite sides of the membrane. There are many different enzyme complexes that are part of redox loops and their catalysed redox reactions can be either electrogenic, electroneutral (non-proton motive) or even pmf-consuming. This article gives conceptual classification of different operational organisations of redox loops and uses this as a platform from which to explore the biodiversity of quinone/quinol-cycling redox systems.     

Protonmotive cooperativity in cytochrome c oxidase

Cooperative linkage of solute binding at separate binding sites in allosteric proteins is an important functional attribute of soluble and membrane bound hemoproteins. Analysis of proton/electron coupling at the four redox centers, i.e. Cu(A), heme a, heme a(3) and Cu(B), in the purified bovine cytochrome c oxidase in the unliganded, CO-liganded and CN-liganded states is presented. These studies are based on direct measurement of scalar proton translocation associated with oxido-reduction of the metal centers and pH dependence of the midpoint potential of the redox centers. Heme a (and Cu(A)) exhibits a cooperative proton/electron linkage (Bohr effect). Bohr effect seems also to be associated with the oxygen-reduction chemistry at the heme a(3)-Cu(B) binuclear center. Data on electron transfer in cytochrome c oxidase are also presented, which, together with structural data, provide evidence showing the occurrence of direct electron transfer from Cu(A) to the binuclear center in addition to electron transfer via heme a. A survey of structural and functional data showing the essential role of cooperative proton/electron linkage at heme a in the proton pump of cytochrome c oxidase is presented. On the basis of this and related functional and structural information, variants for cooperative mechanisms in the proton pump of the oxidase are examined.     

Proton pumping mechanism and catalytic cycle of cytochrome c oxidase: Coulomb pump model with kinetic gating

Using electrostatic calculations, we have examined the dependence of the protonation state of cytochrome c oxidase from bovine heart on its redox state. Based on these calculations, we propose a possible scheme of redox-linked proton pumping. The scheme involves His291 - one of the ligands of the Cu(B) redox center - which plays the role of the proton loading site (PLS) of the pump. The mechanism of pumping is based on ET reaction between two hemes of the enzyme, which is coupled to a transfer of two protons. Upon ET, the first proton (fast reaction) is transferred to the PLS (His291), while subsequent transfer of the second "chemical" proton to the binuclear center (slow reaction) is accompanied by the ejection of the first (pumped) proton. Within the proposed model, we discuss the catalytic cycle of the enzyme.     

The mechanism of coupling between oxido‐reduction and proton translocation in respiratory chain enzymes

The respiratory chain of mitochondria and bacteria is made up of a set of membrane‐associated enzyme complexes which catalyse sequential, stepwise transfer of reducing equivalents from substrates to oxygen and convert redox energy into a transmembrane protonmotive force (PMF) by proton translocation from a negative (N) to a positive (P) aqueous phase separated by the coupling membrane. There are three basic mechanisms by which a membrane‐associated redox enzyme can generate a PMF. These are membrane anisotropic arrangement of the primary redox catalysis with: (i) vectorial electron transfer by redox metal centres from the P to the N side of the membrane; (ii) hydrogen transfer by movement of quinones across the membrane, from a reduction site at the N side to an oxidation site at the P side; (iii) a different type of mechanism based on co‐operative allosteric linkage between electron transfer at the metal redox centres and transmembrane electrogenic proton translocation by apoproteins. The results of advanced experimental and theoretical analyses and in particular X‐ray crystallography show that these three mechanisms contribute differently to the protonmotive activity of cytochrome c oxidase, ubiquinone‐cytochrome c oxidoreductase and NADH‐ubiquinone oxidoreductase of the respiratory chain. This review considers the main features, recent experimental advances and still unresolved problems in the molecular/atomic mechanism of coupling between the transfer of reducing equivalents and proton translocation in these three protonmotive redox complexes.     

Functional implications from an unexpected position of the 49-kDa subunit of NADH:ubiquinone oxidoreductase

Membrane-bound complex I (NADH:ubiquinone oxidoreductase) of the respiratory chain is considered the main site of mitochondrial radical formation and plays a major role in many mitochondrial pathologies. Structural information is scarce for complex I, and its molecular mechanism is not known. Recently, the 49-kDa subunit has been identified as part of the "catalytic core" conferring ubiquinone reduction by complex I. We found that the position of the 49-kDa subunit is clearly separated from the membrane part of complex I, suggesting an indirect mechanism of proton translocation. This contradicts all hypothetical mechanisms discussed in the field that link proton translocation directly to redox events and suggests an indirect mechanism of proton pumping by redox-driven conformational energy transfer.     

The effect of specific antibodies on oxygen uptake and H+ pumping by cytochrome c oxidase vesicles

Antibodies to solubilized cytochrome c oxidase and to subunit III were incubated with liposomal oxidase. In oxygen uptake experiments, the inhibiting effects on RCI of anti-oxidase (primarily anti- subunits II and IV) and anti-III were by different mechanisms: the former, by inhibiting the uncoupled rate; the later, by stimulating the coupled rate. In experiments with H+ translocation, anti-oxidase was without effect, while anti-III was a potent inhibitor of proton pumping. These results are conclusive evidence for redox-linked proton extrusion from the vesicles by the oxidase (and its subunit III).     

On the Mechanism of Proton Translocation by Respiratory Enzyme

The protonmotive function of the respiratory heme-copper oxidases is often described as the sum of two separate mechanisms: a proton pump plus an incomplete Mitchellian redox loop. However, these two functions may be mechanistically intertwined so that the uptake of protons to form water during the reduction of O₂ is a crucial part of the proton pump mechanism itself. This principle can be deduced from thermodynamic, kinetic, mechanistic, as well as from structural considerations, and was first proposed in conjunction with a histidine cycle model of proton translocation [Morgan, J. E., Verkhovsky, M. I., and Wikström, M. (1994). J. Bioenerg. Biomembr. 26, 599–608]. However, histidine cycle models go much further to suggest chemical details of how this principle might be applied.     

Phase-shift model for the aggregation of amoebae: a computer study.

An evolutionary scheme for the origin of chemiosmotic coupling of redox reactions and ATP synthesis is proposed. It is argued that the primitive heterotroph, which generated ATP by substrate level phosphorylation, used some of this ATP in active proton extrusion to regulate cytoplasmic pH. As fermentation substrates were used up, selection favoured organisms which produced a light-dependent redox pump for proton extrusion. This partly replaced the ATP-dependent proton extrusion, thereby economizing on fermentation substrates. The ATP-requiring mechanism was retained for dark proton extrusion. A further economic advantage would come about if the energy of the light-generated proton gradient were used to reverse the ATP-dependent proton pump, leading to chemiosmotic photophosphorylation. This hypothesis explains the origin of the two kinds of proton pump, and their occurrence in the same membrane; the origin of these two prerequisites of chemiosmotic coupling had not previously been adequately explained. The success of the proton pump based on redox loops of alternating vectorial electron and hydrogen atom carriers, rather than the apparently simpler light-driven proton pump of Halobacterium is explained in terms of the ease of converting the former type of cyclic photophosphorylation, but not the latter, into a system bringing about net redox reactions.     

The Redox-Bohr group associated with iron-sulfur cluster N2 of complex I

Proton pumping respiratory complex I (NADH:ubiquinone oxidoreductase) is a major component of the oxidative phosphorylation system in mitochondria and many bacteria. In mammalian cells it provides 40% of the proton motive force needed to make ATP. Defects in this giant and most complicated membrane-bound enzyme cause numerous human disorders. Yet the mechanism of complex I is still elusive. A group exhibiting redox-linked protonation that is associated with iron-sulfur cluster N2 of complex I has been proposed to act as a central component of the proton pumping machinery. Here we show that a histidine in the 49-kDa subunit that resides near iron-sulfur cluster N2 confers this redox-Bohr effect. Mutating this residue to methionine in complex I from Yarrowia lipolytica resulted in a marked shift of the redox midpoint potential of iron-sulfur cluster N2 to the negative and abolished the redox-Bohr effect. However, the mutation did not significantly affect the catalytic activity of complex I and protons were pumped with an unchanged stoichiometry of 4 H(+)/2e(-). This finding has significant implications on the discussion about possible proton pumping mechanism for complex I.     

Proton translocation by transhydrogenase

Transhydrogenase, in animal mitochondria and bacteria, couples hydride transfer between NADH and NADP(+) to proton translocation across a membrane. Within the protein, the redox reaction occurs at some distance from the proton translocation pathway and coupling is achieved through conformational changes. In an 'open' conformation of transhydrogenase, in which substrate nucleotides bind and product nucleotides dissociate, the dihydronicotinamide and nicotinamide rings are held apart to block hydride transfer; in an 'occluded' conformation, they are moved into apposition to permit the redox chemistry. In the two monomers of transhydrogenase, there is a reciprocating, out-of-phase alternation of these conformations during turnover.     

The histidine cycle: A new model for proton translocation in the respiratory heme-copper oxidases

A model of redox-linked proton translocation is presented for the terminal heme-copper oxidases. The new model, which is distinct both in principle and in detail from previously suggested mechanisms, is introduced in a historical perspective and outlined first as a set of general principles, and then as a more detailed chemical mechanism, adapted to what is known about the chemistry of dioxygen reduction in this family of enzymes. The model postulates a direct mechanistic role in proton-pumping of the oxygenous ligand on the iron in the binuclear heme-copper site through an electrostatic nonbonding interaction between this ligand and the doubly protonated imidazolium group of a conserved histidine residue nearby. In the model this histidine residue cycles between imidazolium and imidazolate states translocating two protons per event, the imidazolate state stabilized by bonding to the copper in the site. The model also suggests a key role in proton translocation for those protons that are taken up in reduction of O₂ to water, in that their uptake to the oxygenous ligand unlatches the electrostatically stabilized imidazolium residue and promotes proton release.