Successful theory development in biology: A consideration of the theories of oxidative phosphorylation proposed by Davies and Krebs,Williams and Mitchell

The chemiosmotic theory is normally attributed to Peter Mitchell's formulation published in Nature in 1961. However, the essential elements of the theory were published 9 years earlier by Davies and Krebs. Why, then, was this earlier formulation overlooked? The success of Mitchell's theory is examined in comparison with those of Davies and Krebs and of Williams.     

On the role of the membrane proton conductance in the relationship between rate of respiration and protonmotive force.

The rate of respiration in mitochondria is not a unique function of the protonmotive force, depending on whether the protonmotive force is varied by addition of ADP or uncouplers. This result has been generally considered to contradict the chemiosmotic theory. Recently, O'Shea & Chappell [Biochem. J. (1984) 219, 401-404] claimed that this observation can be reconciled with the chemiosmotic theory, provided only that the proton conductance of the membrane is different in the presence of ADP or uncouplers. This hypothesis is shown here to be necessary but not sufficient to account for the experimental data and the reason for the contradiction between this recent interpretation and earlier interpretations is pointed out.     

Energy transduction function of the quinone reactions in cytochrome bc complexes.

Cytochrome bc1, a multi-subunit integral membrane protein complex found in mammalian mitochondria, plays a central role in the transfer of electrons and protons generated by the oxidation of ubiquinol. According to the classical chemiosmotic theory, quinones shuttle protons across the hydrophobic membrane bilayer with the net result of H+ transfer to the aqueous side and generation of an electrochemical proton gradient. Recently, high-resolution structures of the mitochondrial bc1 complex showed quinone binding sites at one of the transmembrane helices of cytochrome b, and two potentially protonatable histidine residues on the Rieske iron-sulfur protein. The modern biochemical refinements of the original chemiosmotic theory require electron and proton transfer from quinones to particular residues/redox centers of integral membrane proteins and subsequent transfer of H+ to the bulk aqueous phase outside the membrane.     

Transmembrane electron transport and the neutral theory of evolution

Based on the concept of "pairs of basic functional states" the evolution of the first chemiosmotic mechanism of energy conversion is discussed in terms of point mutations, gene duplications and of the neutral theory of evolution. A model for estimating the overall probability of the evolutionary step in question is presented, both for the "selectionist" and "neutralist" position. It is concluded that, concerning the present stage of knowledge, the evolution of transmembrane electron transport is an unsolved problem in evolutionary biology.     

Absence of a unique relationship between active transport of lactose and protonmotive force in E. coli

The relationship between active transport of lactose via the lactose permease and the protonmotive force has been determined in E. coli cells using either the respiratory chain inhibitor cyanide or protonophores to decrease the protonmotive force progressively. In contradiction with the prediction of the delocalized chemiosmotic theory, two different relationships were obtained depending on the method used.     

The Philosophical Origins of Mitchell's Chemiosmotic Concepts

Mitchell's formulation of the chemiosmotic theory of oxidative phosphorylation in 1961 lacked any experimental support for its three central postulates. The path by which Mitchell reached this theory is explored. A major factor was the role of Mitchell's philosophical system conceived in his student days at Cambridge. This system appears to have become a tacit influence on his work in the sense that Polanyi understood all knowledge to be generated by an interaction between tacit and explicit knowing. Early in his life Mitchell had evolved a simple philosophy based on fluctoids, fluctids and statids which was developed in a thesis submitted for the z at the University of Cambridge, England. This aspect of his work was rejected by the examiners and became a tacit element in his intellectual development. It is argued from his various publications that this philosophy can be traced as an underlying theme behind much of Mitchell's theoretical writing in the 50's leading, through his notion of vectorial metabolism, to the formulation and amplification of the chemiosmotic theory in the sixties. This philosophy formed the basis for Mitchell of his understanding of biological systems and gave him his unique approach to cell biology. This revised version was published online in July 2006 with corrections to the Cover Date.     

Potassium-hydrogen ion exchange across barley roots: an introduction to chemiosmotic principles of solute transport

The chemiosmotic theories of ATP synthesis and coupled solute transport across membranes are outlined. A simple experimental system for measuring H⁺ transport and associated cation fluxes across root cortical cells of intact barley plants is described. This system may be explored in a variety of projects or laboratory exercises designed to introduce chemiosmotic principles.     

On the origin of the limited control of mitochondrial respiration by the adenine nucleotide translocator

A thermodynamic control theory previously developed has been applied to mitochondrial oxidative phosphorylation with emphasis on the role of delta microH and coupling and within the paradigm of delocalized chemiosmotic coupling. The basis for the observed distribution of flux control over the participating enzymes is shown to lie in the relative magnitudes of so-called delta microH elasticity coefficients, i.e., the delta microH dependencies of the different mitochondrial processes. In particular the relatively strong delta microH dependence of mitochondrial respiration is responsible for the significant role of the adenine nucleotide translocator in the control of oxidative phosphorylation. Uncoupling decreases the control exerted by this translocator on respiration but increases that exerted on phosphorylation.     

Theory of iron-sulfur center N-2 oxidation and reduction by ATP.

The data of Ohnishi (1975) and of Gutman and coworkers on iron-sulfur center N-2 in mitochondria and submitochondrial particles are examined in as much quantitative detail as possible from the standpoint of both chemiosmotic theory and of chemical intermediate (transductase) theory.A method of examination of the behavior of an energy transduction site by plotting its properties as a function of both the high and low redox potentials on either side of the site is described in some detail.That adding ATP causes center N-2 to go oxidized when buffered redox-wise on the low potential side and reduced when buffered on the high potential side can be explained by both chemiosmotic and chemical intermediate theory.Chemiosmotic explanations consistent with the data exclude location of N-2 at the inside of the mitochondrial membrane, but location at the out side or the middle or mobile across the membrane cannot be ruled out by present data.All four abridged transductase models of chemical intermediate theory can be fitted to the data by choice of parameters.That center N-2 is a simple redox couple located at either side of energy transduction site 1 is ruled out.Further experiments needed to clarify present ambiguities are shown to be: (i) Adding ATP while buffering (redox-wise) the NAD⁺-NADH inside whole mitochondria; (ii) mapping the apparent midpoint potential or, alternatively, the redox state as a complete function of the redox potentials on both the high and low sides; (iii) determination of differences that may be caused by sidedness of the preparation (mitochondria or submitochondrial particles); and (iv) determining effects of changing the partitioning of the proton motive force between ΔpH and membrane potential.     

Proton translocation induced by ATPase activity in chloroplasts

Proton uptake by chloroplasts was induced by light-triggered ATPase activity. A quotient of two was obtained when the initial rate of proton uptake was divided by the rate of P(i) released from ATP. Gramicidin accelerated the rate of ATPase activity and reduced the H(+)/P(i) ratio to 1.4. The results were found to be consistent with the chemiosmotic theory.     

The effect of the energy state of mitochondria on the kinetics of unidirectional cation fluxes

Unidirectional fluxes of triphenylmethylphosphonium and of Cs⁺ as its valinomycin complex were studied using trace concentrations of the cations. The rate constants of influx and efflux were estimated mainly at 0 °C from the uptake kinetics in respiring mitochondria and the in/out ratios in the steady state. The efflux rate constants in the energized state were also measured after dilution of the mitochondrial suspension in the steady state, and in deenergized mitochondria from the efflux rates of cations after inhibition of respiration. It was found that the energy state of mitochondria had little effect on the rate constants of efflux, while the rate of influx was strongly stimulated by respiration. The former finding is not readily explained by the classical chemiosmotic theory, since a transmembrane potential, negative on the inside, formed on energization would be expected to strongly inhibit the efflux of cations. The data may be explained by a pump-and-leak model in which localized electrical fields in hydrophobic domains of the membrane are coupled to the pumping of hydrophobic cations against an electrochemical gradient, while leaks would effect efflux.     

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.     

Mitochondria in malaria and related parasites: ancient,diverse and streamlined

Parasitic organisms have emerged from nearly every corner of the eukaryotic kingdom and hence display tremendous diversity of form and function. This diversity extends to their mitochondria and mitochondrion-derived organelles. While the principles of the chemiosmotic theory apply to all these pathogens, the differences from their hosts provide opportunities for therapeutic development. In this review we discuss examples of mitochondrial systems from a deep-branching phylum, Apicomplexa. Many important human pathogens, such as malaria parasites, belong to this phylum. Unique features of their mitochondria are validated targets for drugs that are selectively toxic to the parasites.     

Analysis of mechanisms of free-energy coupling and uncoupling by inhibitor titrations: theory, computer modeling and experiments

The rates of ATP synthesis and of ATP-driven NAD reduction have been measured in bovine heart submitochondrial particles as a function of the fraction of inhibited redox pumps (in titrations with either antimycin or rotenone) and of the fraction of inhibited ATPases (in titrations with DCCD). The flux control coefficients of the redox and ATPase proton pumps on the rates of ATP synthesis and of ATP-driven NAD reduction have been derived and found to be equal to 1 for both pumps; i.e., both pumps appear to be 'completely rate limiting'. A theoretical analysis of the inhibitor titration approach based on kinetic models of chemiosmotic coupling and on the theory of metabolic control is presented. This analysis (i) shows that the results of the single inhibitor titrations are incompatible with a delocalized chemiosmotic mechanism of energy coupling if the proton conductance of the membrane is sufficiently low with respect to the conductances of the pumps; and (ii) suggests an experimental approach based on the determination of the P/O and the respiratory control ratios at different degrees of inhibition of the proton pumps to establish the origin of the 'loose coupling' of submitochondrial particle preparations. Three independent types of observation show that the 'loose coupling' of the particle preparation is not mainly due to an increased membrane proton conductance. The same and other independent observations are consistent with the view that the loose coupling of submitochondrial particle preparation is due mainly to inhomogeneity, i.e. to the presence of a subpopulation of highly leaky non-phosphorylating vesicles respiring at maximal rate. The results as a whole together with the simulations and analysis presented lead to the conclusion that the mechanism of free-energy coupling in submitochondrial particles is not completely delocalized.     

Surface-mediated proton-transfer reactions in membrane-bound proteins

As outlined by Peter Mitchell in the chemiosmotic theory, an intermediate in energy conversion in biological systems is a proton electrochemical potential difference ("proton gradient") across a membrane, generated by membrane-bound protein complexes. These protein complexes accommodate proton-transfer pathways through which protons are conducted. In this review, we focus specifically on the role of the protein-membrane surface and the surface-bulk water interface in the dynamics of proton delivery to these proton-transfer pathways. The general mechanisms are illustrated by experimental results from studies of bacterial photosynthetic reaction centres (RCs) and cytochrome c oxidase (CcO).     

Energy coupling in the active transport of amino acids by bacteriohodopsin-containing cells of Halobacterium holobium.

Growth of Halobacterium halobium under illumination with limiting aeration induces bacteriorhodopsin formation and renders the cells capable of photophosphorylation. Cells depleted of endogenous reserves by a starvation treatment were used to investigate the means by which energy is coupled to the active transport of [14C]proline, -leucine, and -histidine. Proline was readily accumulated by irradiated cells under anaerobiosis even when the photophosphorylation was abolished by the adenosine triphosphatase inhibitor N,N'-dicyclohexylcarbodimiide (DCCD). The uptake of proline in the dark was limited except when the cells were allowed to accumulate adenosine 5'-triphosphate (ATP) by prior light exposure or by the oxidation of glycerol. DCCD inhibited this dark uptake. These findings essentially support Mitchell's chemiosmotic theory of active transport. The driving force is apparently the proton-motive force developed when protons are extruded from irradiated bacteriorhodopsin or by the dydrolysis of ATP by membrane adenosine triphosphatase. Carbonylcyanide m-chlorophenylhydrazone (CCCP), a proton permeant known to abolish membrane potential, was a strong inhibitor of proline uptake. Leucine transport was also apparently driven by proton-motive force, although its kinetic properties differed from the proline system. Histidine transport is apparently not a chemiosmotic system. Dark- or light-exposed cells show comparable initial rats of histidine uptake, and these processes were only partially inhibited by DCCD or CCCP. The histidine system apparently does not utilize ATP per se since comparable rates of uptake were exhibited by cells of differing intracellular ATP levels. Irradiated cells did effect a greater total accumulation of histidine than dark-exposed cells. These findings suggest that ATP is needed for sustained transport.     

The osmochemistry of electron-transfer complexes

Detailed molecular mechanisms of electron transfer-driven translocation of ions and of the generation of electric fields across biological membranes are beginning to emerge. The ideas inherent in the early formulations of the chemiosmotic hypothesis have provided the framework for this understanding and have also been seminal in promoting many of the experimental approaches which have been successfully used. This article is an attempt to review present understanding of the structures and mechanisms of several osmoenzymes of central importance and to identify and define the underlying features which might be of general relevance to the study of chemiosmotic devices.     

Characterization of a Chinese hamster ovary cell line resistant to uncouplers

The chemiosmotic theory of oxidative phosphorylation and the action of uncouplers was examined by characterizing a clone, UH5, of Chinese hamster ovary (CHO TK-) cells resistant to 5-chloro-3-tert-butyl-2'-chloro-4'-nitrosalicylanilide (S-13), a potent uncoupler of oxidative phosphorylation. About 9-times and 4-times more S-13 was required to effect growth and respiration respectively of UH5 cells compared to the parental CHO TK- cells. UH5 cells were cross-resistant to the uncouplers SF-6847 (3,5-di-tert-butyl-4-hydroxy-benzylidenemalononitrile), carbonylcyanide p-trifluoromethoxyphenylhydrazone and 2,4-dinitrophenol but not to oligomycin, venturicidin or Tevenel. Size, chromosome number and DNA content indicated that the UH5 cell line was probably pseudotetraploid compared to the parental pseudodiploid CHO TK- cells. Hybrid and cybrid cells formed from crosses of UH5 cells and cytoplasts, respectively, with an uncoupler-sensitive cell line were sensitive to S-13 indicating that resistance is probably nuclear-determined. UH5 cell mitochondria had increased cytochrome oxidase and decreased H+-ATPase activities. A fivefold resistance of oxidative phosphorylation to uncouplers was found at the mitochondrial level with respiration driven by either succinate or ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine. In contrast, no difference in sensitivity was found to valinomycin between mitochondria from UH5 and CHO TK- cells. The oligomycin-sensitive H+-ATPase activity of UH5 and CHO TK- cell mitochondria was equally stimulated by the uncoupler S-13. Uncoupler-resistant mitochondria would not be expected on the basis of the chemiosmotic theory, and the relation of the results to other modes of coupling is considered.     

Models of localized energy coupling

It is proven that any model of localized protonmotive energy coupling that relies upon properties of a homogeneous surface phase must, when operated in the steady state, lead to bulk phase electrochemical potentials for protons that are as large as those required by the delocalized chemiosmotic theory. To obtain models consistent with experiments supporting localized energy coupling requires some kind of surface heterogeneity for the proton conducting pathways. Two general classes of heterogeneous surface models are mentioned. One class involves phase-separated lipid domains. The second class involves hydrogen-bonded chains in proteins that traverse the membrane laterally.     

The Chemical Basis of Membrane Bioenergetics

All organisms rely on chemiosmotic membrane systems for energy transduction; the great variety of participating proteins and pathways can be reduced to a few universal principles of operation. This chemical basis of bioenergetics is reviewed with respect to the origin and early evolution of life. For several of the cofactors which play important roles in bioenergetic reactions, plausible prebiotic sources have been proposed, and it seems likely that these cofactors were present before elaborate protein structures. In particular, the hydrophobic quinones require only a membrane-enclosed compartment to yield a minimum chemiosmotic system, since they can couple electron transport and proton translocation in a simple way. It is argued that the central features of modern bioenergetics, such as the coupling of redox reactions and ion translocation at the cytoplasmic membrane, probably are ancient features which arose early during the process of biogenesis. The notion of a thermophile root of the universal phylogenetic tree has been discussed controversially, nevertheless, thermophiles are interesting model organisms for reconstructing the origin of chemiosmotic systems, since they are often acidophiles and anaerobic respirers exploiting iron–sulfur chemistry. This perspective can help to explain the prominent role of iron–sulfur proteins in extant biochemistry as well as the origin of both respiration and proton extrusion within the context of a possible origin of life in the vicinity of hot vents. Received: 6 June 2001 / Accepted: 16 October 2001