Mechanism Of Energy Transduction Research Articles

Cross-bridge states in invertebrate muscles.

Arguments are presented for doubting whether the effect of AMPPNP on insect flight muscle in rigor signals a reversion of the power stroke of attached cross-bridges. Instead, the effect of this nucleotide on insect and other muscles may be better explained in terms of the behavior of detached bridges. Knowledge of events in the detached half of the contractile cycle may nevertheless be relevant to understanding the mechanism of energy transduction.

Mechanism of active transport: free energy dissipation and free energy transduction.

The thermodynamic pathway for "chemiosmotic" free energy transduction in active transport is discussed with an ATP-driven Ca2+ pump as an illustrative example. Two innovations are made in the analysis. (i) Free energy dissipated as heat is rigorously excluded from overall free energy bookkeeping by focusing on the dynamic equilibrium state of the chemiosmotic process. (ii) Separate chemical potential terms for free energy donor and transported ions are used to keep track of the thermodynamic state of each substrate through the reaction cycle. These procedures clarify the mechanism of free energy transduction, even without step-by-step analysis. The results show that free energy exchange must occur in its entirety among protein-bound species. Imposition of conditions for an adequate rate of physiological function further indicates (i) that the standard free energy of hydrolysis of protein-bound ATP (to yield protein-bound products) needs to differ substantially from the standard free energy of hydrolysis in solution and (ii) that binding sites for the transported ions must have different affinities when facing opposite sides of the membrane. The results also demonstrate that step-by-step "basic" free energy changes (often used in the form of free energy level diagrams) are inherently unsuited for analysis of the mechanism of free energy transduction.

521—Light-induced redox reactions in pigmented BLM

This paper is concerned with light-mediated phenomena in membranes of photosynthesis and vision and their in vitro model bilayer lipid membranes (BLM) of planar configuration containing appropriate photoactive compounds. Chloroplast extract BLM, bovine rhodopsin BLM and bacteriorhodopsin BLM are used as examples. Particular emphasis is placed on those molecular mechanisms of photoelectrochemical energy transduction in these pigmented lipid bilayers, which are relevant for the elucidation of photosynthetic and visual processes. Additionally, a pigmented BLM separating two aqueous solutions containing redox couples has been likened to that of a double Schottky barrier cell.

Light-induced redox reactions in pigmented BLM

This paper is concerned with light-mediated phenomena in membranes of photosynthesis and vision and their in vitro model bilayer lipid membranes (BLM) of planar configuration containing appropriate photoactive compounds. Chloroplast extract BLM, bovine rhodopsin BLM and bacteriorhodo0psin BLM are used as examples. Particular emphasis is placed on those molecular mechanisms of photoelectrochemical energy transduction in these pigmented lipid bilayers, which are relevant for the elucidation of photosynthetic and visual processes. Additionally, a pigmented BLM separating two aqueous solutions containing redox couples has been likened to that of a double Schottky barrier cell.

Disruptiin of energy transductiin in sarcoplasmic reticulum by trypsin cleavage of (Ca2+ + Mg2+)-ATPase.

Proteolytic digestion of sarcoplasmic reticulum vesicles with trypsin has been used as a structural modification with which to examine the interaction between the ATP hydrolysis site and calcium transport sites of the (Ca2++Mg2+)-ATPase. The kinetics of trypsin fragmentation were examined and the time course of fragment production compared with ATP hydrolytic and calcium uptake activities of the digested vesicles. The initial cleavage (TD 1) of the native ATPase to A and B peptides has no effect on the functional integrity of the enzyme, hydrolytic and transport activities remaining at the levels of the undigested control. Concomitant with the second tryptic cleavage (TD 2) of the A peptide to A1 and A2 fragments, calcium transport is inhibited. Kinetic analysis demonstrates that the rate constant for inhibition of calcium uptake is correlated with the rate constant of a fragment disappearance. Both Ca2+-dependent and total ATPase activities are unaffected by this second cleavage. Passive loading of vesicles with calcium and subsequent efflux measurements show that transport inhibition is not due to increased permeability of the membrane to calcium even at substantial extents of digestion. Steady-state levels of acidstable phosphoenzyme are unaffected by either TD 1 or TD 2, indicating that uncoupling of the hydrolytic and transport functions does not increase the turnover rate of the enzyme and that TD 2 does not change the essential characteristics of the ATP hydrolysis site. Sarcoplasmic reticulum (SR) vesicles were examined for the presence of “tightly bound” nucleotides and are shown to contain 2.8–3.0 nmol ATP and 2.6–2.7 nmol ADP per mg SR protein. The ADP content of SR remains essentially unchanged with TD 1 cleavage of the ATPase enzyme to A and B peptides, but declines upon TD 2 in parallel with the digestion of the A fragment and the loss of calcium uptake activity of the vesicles. The ATP content is essentially constant throughout the course of trypsin digestion. The results are discussed in terms of current models of the SR calcium pump and the molecular mechanism of energy transduction.

Electron and proton transfers in chemical and biological quinone systems

Some redox chemistry and biochemistry of p-benzoquinones are summarised. In particular, the behaviour of quinols and quinones at electrode surfaces and in solution, together with the mechanism of reduction of soluble cytochrome c by quinols, produces a model of electron transfer relevant to biological systems. In such a model the biological transfers occur by collisional reactions of the quinone and quinol with the protein donors and acceptors. After collision, the detailed reactions occur via bound enzyme intermediates but electronic mobility between the donors and acceptors is provided by diffusional mobility of the quinone molecules. Such considerations have allowed a detailed investigation of the redox reactions of quinols with a complex biological multiprotein system, the bc complex. A probable mechanism of electron transfer into and through this complex is presented, together with some discussion of the mechanism of energy transduction which would operate in the intact biological system.

Ecto-ATPase.

An ecto-adenosine triphosphatase (E.C. 3.6.1.4 ATP-phosphohydrolase) is shown to be localized on the outer surface of varieties of cell membrane. The enzyme is different from the ATPase involved in biological energy transduction and ion transport mechanism. The characteristic of the enzyme lies in having a very broad substrate specificity and is inhibited by EDTA and higher concentration of ATP. The enzyme is dependent on bivalent metal ions, Mg++ or Ca++ for its optimum activity. The enzyme is highly sensitive to SH-reagents but insensitive to inhibitors of mitochondrial ATPase or Na+- K+- ATPase. The possible functions of the enzyme in being oriented outside the cell membrane is discussed.

Functional reassembly of membrane proteins in planar lipid bilayers.

Recent progress in membrane biology has brought us to a stage where it is possible to associate complex biological processes to identifiable membrane proteins. Technical advances in the biochemical characterization and purification of membrane proteins have contributed a wealth of structural information. The reconstitution approach has proved to be valuable in our efforts to understand the molecular mechanisms of membrane transport and energy transduction.

Mechanisms of Energy Transduction in Plant Photosynthesis: ESR, ENDOR and MOs of the Primary Acceptors

AbstractComparison of the optical, paramagnetic and redox properties of the reduced primary acceptors of Photosystems (PS) I and II with those of chlorophyll and pheophytin anions suggests that chlorophyll is the primary reduced product in PS I and pheophytin in PS II. Molecular orbital calculations provide a description of the electronic profiles of the radicals, and indicate that the protein environment of the chromophores in the reaction center may impose specific orientations and induce hydrogen bonding to some of the chlorophyll substituent groups. Examination of all the primary acceptors postulated for green plants and purple bacteria suggests that (bacterio)chlorophyll‐like acceptors may be obligatory to effect the rapid primary charge separation, because they allow favorable orbital overlap between donor and acceptor. Extrapolation of these results to green bacteria suggests that bacteriochlorophyll a and bacteriopheophytin c may act as transient electron acceptors in these organisms.

Chemical potential of bound ligand, an important parameter for free energy transduction

The chemical potential (mu(L,b)) of a ligand L bound to a protein or enzyme can be rigorously defined, and this paper describes some of its properties in relation to other thermodynamic parameters, with emphasis on thermodynamic parameters that may be used in the elucidation of the mechanism of biological free energy transduction. Free energy transduction involves the transfer of free energy from one molecule to another, and the actual transfer may often occur while both molecules are bound to the transducer enzyme, which means that mu(L,b) for one bound ligand increases at the expense of mu(L,b) for the other. The free energy change for the overall reaction may be very small, and it is not possible to express the phenomenon of transfer, in thermodynamic terms, without the explicit use of mu(L,b) as a parameter.

Soluble and membrane-bound paramagnetic centers in Methanobacterium Bryantii

1. Introduction The methanogenic bacteria are members of a unique group of organisms (denoted Archaebacteria) which are apparently only distantly related to prokaryotes and eukaryotes [ 1,2]. Methanogens are characterized by the ability to grow autotrophically solely by the reduction of COZ by Hz to produce methane. Several electron carriers and ‘factors’ unique to methanogens have been isolated, including coenzymes Fd2,, [3,4], FM2 [5] and F4% [5], as well as coenzyme M (Zmercaptoethanesulfonic acid [6,4]). While some light has been shed on the roles of FJ2,, and coenzyme M in COZ reduction [4,7], virtu- ally nothing is known about the mechanism(s) of energy transduction via electron transport from Hz to COZ. By using low-temperature EPR spectroscopy I report here the presence of oxidized and reduced paramagnetic species,. both soluble and membrane- bound, in M.

The orientation of the substrate sites of formate dehydrogenase and fumarate reductase in the membrane of Vibrio succinogenes

Formate dehydrogenase and fumarate reductase are involved in the electron transport phosphorylation system of Vibrio succinogenes. The orientation of the active sites of these enzymes in the cytoplasmic membrane of the bacterium was investigated with the aim of elucidating the mechanism of energy transduction. This was done by measuring the accessibilities of the enzymes to substrates, dyes and inhibitors both in cells and in cell-derived particles obtained with the French press. 1. 1. After treatment of the cells with lysozyme and EDTA, followed by fractionation, both enzymes were found exclusively in the membranous fractions, while the periplasmic as well as the cytoplasmic fractions were devoid of both of the enzymic activities. 2. 2. The sites of dye interaction of fumarate reductase were inaccessible to non-permeant dyes in cells, but were fully accessible in French-press particles. The K m for succinate as measured with the permeant methylene blue as acceptor was increased ten fold on lysis of the cells. The K m measured in the particles was similar to that of lysed cells and was not altered by lysis. 3. 3. The rates of formate oxidation in the presence of non-permeant dyes and the K m for formate were unaffected by cell lysis. On lysis of French-press particles, formate oxidation with both permeant and non-permeant acceptors was increased about three fold. The extent of stimulation was not altered by inhibition of the enzymic activities. 4. 4. Succinate oxidation by particles was fully inhibited by 4-chloromercuriphenyl sulfonate whereas that of cells was fully resistant. Formate dehydrogenase in cells was inhibited by 4-diazophenyl sulfonate when added together with formate. This compound also inhibited the enzyme in the particles when given in the absence of a lytic agent or after its addition. However, most of the enzyme remained active if the inhibitor was added before the lytic agent. 5. 5. Fumarate and succinate were actively taken up by the cells from the medium, while formate did not even penetrate through the membrane of the bacteria. 6. 6. It is concluded that the substrate and the dye-reactive sites of formate dehydrogenase face the outside, while those of fumarate reductase face the inside of the cytoplasmic membrane of cells of V. succinogenes. About 70% of the French-press particles were inverted with respect to the substrate and dye sites of the enzymes. The membrane is impermeable to formate and does not contain a specific transporter. The electrogenic liberation of protons on the outside and the uptake of protons from the cytoplasm of the bacteria, which accompany electron transport, can be explained on the basis of the orientation of the substrate sites of the enzymes without net transport of protons across the membrane.

Energy-transducing proteins in thermophilic biomembranes.

Biomembranes are the major site of energy transduction. The chemisomotic theroy of energy transduction is based on the following four major systems (i) H+-ATPase which is composed of a catalytic portion (F1) and a H+-channel (Fo), (ii) electron transport components, (iii) H+-linked porters, and (iv) a H+-impermeable lipid bilayer which is plugged through by systems i to iii that are specially oriented to translocate H+. Studies on the molecular mechanism of energy transduction have been hampered by the impurity, instability and complexity of preparations of membrane proteins from mesophilic organism. However, using stable, simple membrane proteins from a thermophilic bacterium, we obtained the following results: 1) Thermophilic H+-ATPase was dissociated into 5 subunits of F1 and 3 subunits of Fo and their functions and structures were studied by reconstitution. F1 was crystallized. 2) Thermophilic cytochrome oxidase, cytochrome c and NADH-dehydrogenase were purified. In contrast to the complex mitochondrial cytochrome oxidase (7 subunits) and NADH-dehydrogenase (3 subunits), the purified thermophilic proteins were shown to be composed of single components. 3) H+-linked porters such as a H+-driven amino acid carrier and a Na+-H+ antiporter were characterized. 4) Thermophilic lipids were shown to be completely saturated. Using these stable lipids, liposomes capable of H+-driven vectorial reactions including net ATP synthesis and alanine transport were reconstituted.

Molecular mechanism of protein biosynthesis and an approach to the mechanism of energy transduction.

In 1941 Fritz Lipmann wrote an article in Advances in Enzymology which is now regarded as a landmark in the history of biochemistry. In this review he contemplated his new concept that the phosphate bonds serve as general carriers in energy transformation and in biosynthesis. The concept of “phosphate bond energy” or “energy-rich phosphate bond” has promptly prevailed among biochemists, and since then much effort has been focused on the mechanism of “group transfer” reactions in which the phosphate bond energy is very often utilized for the synthesis of covalent bonds.

Uncouplers of oxidation phosphorylation

Numerous compounds, covering a broad range of chemical structures, selectively interfere with the mitochondrial mechanism for transforming energy derived from oxidative metabolism into regeneration of ATP. Within some chemical classes, structure-activity correlations have been useful. Several distinct mechanism of uncoupling have been characterized: 1. (1) trans-membrane proton transport 2. (2) trans-membrane cation transport and exchange 3. (3) modification of essential protein factors of membrane 4. (4) competition with phosphate (trivial case) Others may well exist. Uncouplers can serve as probes of the mitochondrial energy transduction mechanism.

Muscle contraction: a mechanism of energy transduction

A mechanism of muscle contraction is presented in which energy from the hydrolysis of MgATP is transferred directly to conformational strain in a flexible segment of the myosin head. That segment is proximal to both the active site and the subfragment 1—subfragment 2 hinge (the portion of the myosin molecule that connects each of its two enzymatically active globular heads to the long thin helical body). This proximity allows configurational changes at the active site, which are an intrinsic part of the enzymatic mechanism, to impose a localized strain, or distortion, near the hinge. The energy, trapped in the protein this way, is subsequently used for mechanical work when other enzymatically-induced conformational changes free the strained segment of the myosin head to unbend. As this happens, the head rotates and the distal end (opposite the hinge) attaches to the actin filament and pulls on it. In this mechanism, actin interacts with myosin in two different ways: (1) at the active site where it activates a step in the hydrolysis of MgATP that frees the head to rotate; (2) at the distal end of myosin, where it forms the grip through which the rotating head pulls on the actin filament. The first interaction allows actin to initiate primary movement of the myosin head; the second directs the force and allows the movement of the head to be used for the sliding motion of the actin and myosin filaments during contraction. In this model, there are also two different energy transfers: one occurs in the transduction process itself when energy from hydrolysis is trapped as conformational distortion in the hinge region; the other occurs, reversibly, when actin and myosin form and then break the distal grip; in this second transfer there is no net energy change in the course of a cycle. A chemical mechanism is suggested to explain actin-activation of hydrolysis at the active site-hinge region.

Cooperativity in the photocycle of purple membrane of Halobacterium halobium with a mechanism of free energy transduction

Cooperativity in the photocycle of purple membrane of Halobacterium halobium with a mechanism of free energy transduction

Kinetic analysis of light-dependent exchange of adenine nucleotides on chloroplast coupling factor CF 1

1. Introduction Chloroplast coupling factor CF, contains tightly bound ADP which is exchanged against added ADP only on energization of the thylakoid membranes [l-6]. This ADP binding site is probably different from the catalytic ADP binding site in the process of photophosphorylation [S-9]. Nevertheless it may be involved in the mechanism of energy transduction. Light-induced nucleotide exchange and photophos- phorylation show a parallel behavior in their depen- dence on several external factors, e.g., light intensity, pH,uncoupling and electron transport inhibition [lo]. The mechanism of ADP exchange involves two consecutive reversible reactions and three different forms of CFI which can be characterized by different states of the ADP binding site [ 111: (i) A stable CF1 -ADP complex (tightly-bound ADP); (ii) An unstable transitory CFl-‘ADP complex (loosely bound ADP); (iii) A metastable dissociated complex containing an unoccupied ADP binding site. The energy-requiring step is thought to be related to a conformational change of the protein by which tightly bound ADP is transferred to the loosely bound form. The loosely bound ADP molecule can be replaced by dissociation and re-association with a free ADP molecule. The latter reaction does not require an energized state of the membrane [ 111. During illumination of the chloroplasts all three forms *

43] Design of a simple rapid mixing and quenching device and its use for measurement of the kinetics of ATP synthesis by submitochondrial particles

Publisher Summary Submitochondrial particles subjected to an artificially imposed electrochemical gradient of protons catalyzes the net synthesis of ATP, similar to chloroplasts and mitochondria, and prompted a study of the initial kinetics of this process to test the “kinetic competence” of an electrochemical proton gradient as an intermediate in oxidative phosphorylation. Continuous flow mixing with chemical quenching was chosen as an experimental technique so that ATP synthesis could be measured directly by analytical means. However, to keep the amount of submitochondrial particles required for such experiments within reasonable limits, an inexpensive apparatus was designed for mixing relatively small volumes (0.5 ml) of reagents. The chapter discusses some of the implications for the mechanism of ATP synthesis. The results of the experiments presented in the chapter indicated that an electrochemical gradient of protons was kinetically sufficient for being an intermediate in oxidative phosphorylation. Such experiments also describes the utility of the continuous flow technique for studying the mechanism of ATP synthesis and energy transduction and complement the classic spectrophotometric studies of the kinetic properties of the electron transfer components of the respiratory chain.

Large-scale rotational motions of proteins detected by electron paramagnetic resonance and fluorescence

Direct spectroscopic measurements of rotational motions of proteins and large protein segments are crucial to understanding the molecular dynamics of protein function. Fluorescent probes and spin labels attached to proteins have proved to be powerful tools in the study of large-scale protein motions. Fluorescence depolarization and conventional electron paramagnetic resonance (EPR) are applicable to the study of rotational motions in the nanosecond-to-microsecond time range, and have been used to demonstrate segmental flexibility in an antibody and in myosin. Very slow rotational motions, occurring in the microsecond-to-millisecond time range, are particularly important in supramolecular assemblies, where protein motions are restricted by association with other molecules. Saturation transfer spectroscopy (ST-EPR), a recently developed electron paramagnetic resonance (EPR) technique that permits the detection of rotational correlation times as long as 1 ms, has been used to detect large-scale rotational motions of spin-labeled proteins in muscle filaments and in membranes, providing valuable insights into energy transduction mechanisms in these assemblies.