Audrius Jasaitis

Conversion of biomembrane-produced energy into electric form. I. Submitochondrial particles

The hypothesis of an electric membrane potential generated by respiration or ATP hydrolysis in submitochondrial particles has been verified. To this end a number of synthetic ions penetrating lipid membranes were used. Penetrating anions of phenyl dicarbaundecaborane (PCB−), tetraphenyl boron and picrate were shown to accumulate in sonicated submitochondrial particles in an energy-dependent manner. The process was inhibited by rotenone, antimycin and cyanide if supported by respiration, and by oligomycin, if ATP was used as the energy source. Uncouplers were inhibitory in both cases. The following oxidation reactions were found to support the energy-dependent accumulation of PCB−: oxidation of NADH by oxygen or fumarate; oxidation of succinate or ascorbate by oxygen; oxidation of NADPH by NAD+. In the latter case, which is the reverse of the energy-requiring transhydrogenase reaction, ion transport was inhibited by NADH and NADP+ as well as by uncouplers. Oxidation of NADH by NADP+ in the energy-requiring transhydrogenase reaction was accompanied by an efflux of PCB− anions which had accumulated during succinate oxidation. The redox ‘succinate-ferricyanide’ couple could not be used as a supply of energy for the accumulation of PCB− Particles deprived of the coupling factor F1 showed a decreased ability for respiration-dependent anion uptake, the process being stimulated by oligomycin. ATP-driven PCB− accumulation was completely absent in F1-deprived particles but could be reconstituted after preincubation with F1. The active accumulation of anions penetrating into particles was readily distinguished from passive anion absorption, since the latter did not require energy and could be demonstrated both in native particles and in those deprived of F1, as well as in phospholipid micelles. The energy-dependent accumulation of anions penetrating into submitochondrial particles was accompanied by alkalinization of the incubation medium. The efflux of ions upon the cessation of the energy supply induced acidification. Anion accumulation was followed by the suppression of other energy-linked functions of submitochondrial particles. Under the same conditions the penetrating cations, dibenzyl dimethyl ammonium, tetrabutyl ammonium and triphenyl methyl phosphonium, did not affect either the pH of the medium or energy-linked functions. It was concluded that a mechanism for ion accumulation in submitochondrial particles is specific for the sign of the charge but not for other features of the penetrating compounds. This mechanism operates in such a way that anions, but not cations, are pumped into the particle as if the process were supported by an electric field, orientated across the membrane, being positive inside the particles.

Conversion of biomembrane-produced energy into electric form. II. Intact mitochondria.

Abstract The transport of synthetic ions penetrating across intact mitochondrial membranes has been investigated. It is shown that anions of phenyl dicarbaundecaborane (PCB − ) are extruded from mitochondria on transition to the energized state. Discharge of the energized state is accompanied by movement of the extruded anions back into the mitochondria. The penetrating cations dibenzyl dimethyl ammonium (DDA + ), tetrabutyl ammonium and triphenyl methyl phosphonium, when added to liver or heart mitochondria in the presence of oxidizable substrates or ATP, bring about the same responses that accompany the active transport of natural penetrating cations Ca 2+ or K + in the presence of valinomycin, i.e. acidification of the incubation mixture, a transient increase in ATPase and oxidation rate in State 4, cyclic oxidation of NAD(P)H reduced by succinate and swelling of the mitochondrial matrix. The latter process requires the addition of inorganic phosphate. DDA + -induced swelling is found to be supported by both ATP hydrolysis and respiratory chain electron transfer from substrates to oxygen or to ferricyanide. All effects of penetrating cations in mitochondria are potentiated by the addition of small amounts of the penetrating anions, PCB − or tetraphenyl boron, which increase the permeability of the membrane for the cations under study. The data obtained confirm the conclusion that it is the electric field (negative inside the mitochondria) which is the motive force for the transport of penetrating ions across the mitochondrial membrane.

Proton translocation by cytochrome c oxidase.

Cell respiration in mitochondria and some bacteria is catalysed by cytochrome c oxidase, which reduces O2 to water, coupled with translocation of four protons across the mitochondrial or bacterial membrane,,. The enzymes catalytic cycle consists of a reductive phase, in which the oxidized enzyme receives electrons from cytochrome c, and an oxidative phase, in which the reduced enzyme is oxidized by O2. Previous studies indicated that proton translocation is coupled energetically only to the oxidative phase, but this has been challenged. Here, with the purified enzyme inlaid in liposomes, we report time-resolved measurements of membrane potential, which show that half of the electrical charges due to proton-pumping actually cross the membrane during reduction after a preceding oxidative phase. pH measurements confirm that proton translocation also occurs during reduction, but only when immediately preceded by an oxidative phase. We conclude that all the energy for proton translocation is conserved in the enzyme during its oxidation by O2. One half of it is utilized for proton-pumping during oxidation, but the other half is unlatched for this purpose only during re-reduction of the enzyme.

Anilinonaphthalenesulfonate fluorescence changes induced by non-enzymatic generation of membrane potential in mitochondria and submitochondrial particles

Abstract Changes in the fluorescence of 1-anilino-8-naphthalenesulfonate (ANS − ) accompanying non-enzymatic generation of the membrane potential in mitochondria and sonicated submitochondrial particles have been demonstrated. Generation of the membrane potential was induced by addition of an ionophore (valinomycin for K + , or tetrachlorotri-fluoromethylbenzimidazole for H + ) under conditions where there existed K + (or H + ) gradients across the mitochondrial membrane. The ANS − fluorescence decreased when the mitochondrial (or particle) interior became more negative, and increased when it became more positive. Collapse of the membrane potential reversed the ANS − responses. A hypothesis is put forward to explain the energy-dependent ANS − responses in mitochondria and particles by the membrane potential-induced redistribution of ANS − between the membrane and water phases.

Membrane potential generation by two reconstituted mitochondrial systems: Liposomes inlayed with cytochrome oxidase or with ATPase

Abstract Formation of a membrane potential in two types of liposomes, one inlayed with cytochrome c + cytochrome oxidase, and another, with oligomycin-sensitive ATPase, has been demonstrated. To detect a membrane potential, phenyl dicarbaundecaborane (PCB − ), a penetrating anion probe, was used. The first type of liposome was reconstituted from a solution of purified cytochrome oxidase, mitochondrial phospholipids and cytochrome c , the latter being enclosed inside liposomes. Cytochrome c bound to the outer surface of the liposome membrane was removed by washing with NaCl. Such liposomes catalyzed oxidation of ascorbate by oxygen in the presence of phenazine methosulfate or N , N , N ′, N ′-tetramethyl- p -phenylenediamine. The oxidation was found to support the PCB − uptake by liposomes. The PCB − response was prevented and reversed by cyanide, protonophorous uncouplers and external cytochrome c . Liposomes of the second type were prepared from a solution of mitochondrial phospholipids, coupling factors F 1 and F c , and the hydrophobic proteins of the oligomycin-sensitive ATPase. These liposomes catalyzed ATP hydrolysis coupled with the PCB − uptake. The latter effect was prevented and reversed by oligomycin and uncouplers. The conclusion is made that membrane potential can be independently formed by enzymic reactions of two different kinds: (1) redox ( e.g. cytochrome c oxidase) and (2) hydrolytic (ATPase).

Nanosecond electron tunneling between the hemes in cytochrome bo3

Biological electron transfer (eT) between redox-active cofactors is thought to occur by quantum-mechanical tunneling. However, in many cases the observed rate is limited by other reactions coupled to eT, such as proton transfer, conformational changes, or catalytic chemistry at an active site. A prominent example of this phenomenon is the eT between the heme groups of mitochondrial cytochrome c oxidase, which has been reported to take place in several different time domains. The question of whether pure eT tunneling in the nanosecond regime between the heme groups can be observed has been the subject of some experimental controversy. Here, we report direct observations of eT between the heme groups of the quinol oxidase cytochrome bo3 from Escherichia coli, where the reaction is initiated by photolysis of carbon monoxide from heme o3. eT from CO-dissociated ferrous heme o3 to the low-spin ferric heme b takes place at a rate of (1.2 ns)−1 at 20°C as determined by optical spectroscopy. These results establish heme–heme electron tunneling in the bo3 enzyme, a bacterial relative to the mitochondrial cytochrome c oxidase. The properties of eT between the closely lying heme groups in the heme–copper oxidases are discussed in terms of the reorganization energy for the process, and two methods for assessing the rate of electron tunneling are presented.

Ligand Dynamics in an Electron Transfer Protein PICOSECOND GEMINATE RECOMBINATION OF CARBON MONOXIDE TO HEME IN MUTANT FORMS OF CYTOCHROME c

Substitution of the heme coordination residue Met-80 of the electron transport protein yeast iso-1-cytochrome c allows external ligands like CO to bind and thus increase the effective redox potential. This mutation, in principle, turns the protein into a quasi-native photoactivable electron donor. We have studied the kinetic and spectral characteristics of geminate recombination of heme and CO in a series of single M80X (X = Ala, Ser, Asp, Arg) mutants, using femtosecond transient absorption spectroscopy. In these proteins, all geminate recombination occurs on the picosecond and early nanosecond time scale, in a multiphasic manner, in which heme relaxation takes place on the same time scale. The extent of geminate recombination varies from >99% (Ala, Ser) to ∼70% (Arg), the latter value being in principle low enough for electron injection studies. The rates and extent of the CO geminate recombination phases are much higher than in functional ligand-binding proteins like myoglobin, presumably reflecting the rigid and hydrophobic properties of the heme environment, which are optimized for electron transfer. Thus, the dynamics of CO recombination in cytochrome c are a tool for studying the heme pocket, in a similar way as NO in myoglobin. We discuss the differences in the CO kinetics between the mutants in terms of the properties of the heme environment and strategies to enhance the CO escape yield. Experiments on double mutants in which Phe-82 is replaced by Asp or Gly as well as the M80D substitution indicate that such steric changes substantially increase the motional freedom-dissociated CO.

Plasticity of Proton Pathway Structure and Water Coordination in Cytochrome c Oxidase

Cytochrome c oxidase (CytcO) is a redox-driven, membrane-bound proton pump. One of the proton transfer pathways of the enzyme, the D pathway, used for the transfer of both substrate and pumped protons, accommodates a network of hydrogen-bonded water molecules that span the distance between an aspartate (Asp132), near the protein surface, and glutamate Glu286, which is an internal proton donor to the catalytic site. To investigate how changes in the environment around Glu286 affect the mechanism of proton transfer through the pathway, we introduced a non-hydrogen-bonding (Ala) or an acidic residue (Asp) at position Ser197 (S197A or S197D), located ∼7 Å from Glu286. Although Ser197 is hydrogen-bonded to a water molecule that is part of the D pathway “proton wire,” replacement of the Ser by an Ala did not affect the proton transfer rate. In contrast, the S197D mutant CytcO displayed a turnover activity of ∼35% of that of the wild-type CytcO, and the O2 reduction reaction was not linked to proton pumping. Instead, a fraction of the substrate protons was taken from the positive (“incorrect”) side of the membrane. Furthermore, the pH dependence of the proton transfer rate was altered in the mutant CytcO. The results indicate that there is plasticity in the water coordination of the proton pathway, but alteration of the electrostatic potential within the pathway results in uncoupling of the proton translocation machinery.

Subpicosecond oxygen trapping in the heme pocket of the oxygen sensor FixL observed by time-resolved resonance Raman spectroscopy

Dissociation of oxygen from the heme domain of the bacterial oxygen sensor protein FixL constitutes the first step in hypoxia-induced signaling. In the present study, the photodissociation of the heme-O2 bond was used to synchronize this event, and time-resolved resonance Raman (TR3) spectroscopy with subpicosecond time resolution was implemented to characterize the heme configuration of the primary photoproduct. TR3 measurements on heme-oxycomplexes are highly challenging and have not yet been reported. Whereas in all other known six-coordinated heme protein complexes with diatomic ligands, including the oxymyoglobin reported here, heme iron out-of-plane motion (doming) occurs faster than 1 ps after iron–ligand bond breaking; surprisingly, no sizeable doming is observed in the oxycomplex of the Bradyrhizobium japonicum FixL sensor domain (FixLH). This assessment is deduced from the absence of the iron–histidine band around 217 cm−1 as early as 0.5 ps. We suggest that efficient ultrafast oxygen rebinding to the heme occurs on the femtosecond time scale, thus hindering heme doming. Comparing WT oxy-FixLH, mutant proteins FixLH-R220H and FixLH-R220Q, the respective carbonmonoxy-complexes, and oxymyoglobin, we show that a hydrogen bond of the terminal oxygen atom with the residue in position 220 is responsible for the observed behavior; in WT FixL this residue is arginine, crucially implicated in signal transmission. We propose that the rigid O2 configuration imposed by this residue, in combination with the hydrophobic and constrained properties of the distal cavity, keep dissociated oxygen in place. These results uncover the origin of the “oxygen cage” properties of this oxygen sensor protein.

The effect of oncotic pressure on heart muscle mitochondria

Abstract The role of oncotic pressure ( i.e. pressure created by non-penetrants of high molecular weight) in structural responses of mitochondria has been studied. It has been found that treatment of beef of rabbit heart mitochondria by a synthetic non-penetrant of high molecular weight, polyvinyl pyrrolidone, induces a decrease in the intermembrane (intracristal) space and an increase in the matrix space of mitochondria. As a result, the appearance of the in vitro mitochondria proves to be similar to that of the in situ ones. If a Waring blender is used to homogenize the tissue, only a portion of the mitochondria respond to polyvinyl pyrrolidone. If a glass-Teflon homogenizer is used instead all the mitochondria prove responsive. The addition of 0.5 mM polyvinyl pyrrolidone is found to be sufficient for the effect to be observable. In the presence of polyvinyl pyrrolidone, energy-dependent changes in mitochondrial structure can be demonstrated. The increase in matrix space by polyvinyl pyrrolidone treatment enlarges even more when an energy source, a penetrating weak acid and a penetrating cation are added. The size of the matrix increases in the following order: (1) de-energized mitochondria without polyvinyl pyrrolidone, (2) de-energized + polyvinyl pyrrolidone, (3) energized + polyvinyl pyrrolidone, (4) as (3) + phosphate (“twisted” configuration of cristae), (5) as (3) + phosphate + Ca 2+ . Structural changes resembling those indicated in points (2)–(5) are shown for mitochondria in the tissue, when pieces of rat diaphragm muscle treated with an uncoupler, phosphate, and Ca 2+ were studied in conditions excluding anaerobiosis. The effect of polyvinyl pyrrolidone is suggested to be due to it balancing the oncotic pressure created by high molecular weight compounds dissolved in the intermembrane water, which are incapable of penetrating the outer mitochondrial membrane. A concept is discussed considering mitochondrial structure changes as a function of the osmotic gradient across the inner membrane and the oncotic gradient across the outer membrane of mitochondria.