A role for the protein in internal electron transfer to the catalytic center of cytochrome c oxidase

Internal electron transfer (ET) to heme a(3) during anaerobic reduction of oxidized bovine heart cytochrome c oxidase (CcO) was studied under conditions where heme a and Cu(A) were fully reduced by excess hexaamineruthenium. The data show that ET to heme a(3) is controlled by the state of ionization of a single protolytic residue with a pK(a) of 6.5 +/- 0.2. On the basis of the view that ET to the catalytic site is limited by coupled proton transfer, this pK(a) was attributed to Glu60 which is located at the entrance of the proton-conducting K channel on the matrix side of CcO. It is proposed that Glu60 controls proton entry into the channel. However, even with this channel open, there is the second factor that regulates ET, and this is ascribed to the rate of proton diffusion in the channel. In addition, it is concluded that proton transfer in the K channel is reversibly inhibited by the detergent Triton X-100. It is also found that the rate of ET to heme a(3) in the as-isolated resting enzyme and in CcO "activated" by reaction of fully reduced enzyme with O(2) is the same, implying that the catalytic sites of these two forms of oxidized enzyme are essentially identical.     

Site specific interaction of protons liberated from Photosystem II oxidation with a hydrophobic membrane component of the chloroplast membrane

Chloroplast membranes have been shown previously to undergo a change in radioactive labeling by chemical modification reagents that is dependent on electron transport and protolytic events in Photosystem II. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis has been used to show that a low molecular weight chloroplast polypeptide (7.2 Kilodaltons) undergoes the most change in acetic anhydride labeling upon Photosystem II electron transport. A similar polypeptide has been identified by other workers as a component of the hydrophobic trans-membrane proton channel in chloroplasts. Photosystem I electron transport does not give the change in level of incorporation of acetic anhydride into this membrane protein. These results suggest that protons liberated from Photosystem II interact with a hydrophobic portion of the chloroplast membrane, perhaps with the trans-membrane proton channel.     

The electric generator in the photosynthesis of green plants. II. Kinetic correlation between protolytic reactions and redox reactions

In a preceding paper (Junge, W. and Ausländer, W. (1974) Biochim. Biophys. Acta 333, 59–70), we attributed the four protolytic reactions at the outer and the inner side of the functional membrane of photosynthesis to the protolytic properties of the redox components, water, plastoquinone and the terminal acceptor. The experimental evidence presented was conclusive except for one argument. The rate of the protolytic reactions as detected by the dye cresol red after a short flash of light was considerably slower than the rate of the corresponding redox reactions.In this communication it is demonstrated that the rate of proton uptake from the outer phase of the functional membrane is slowed down by a diffusion barrier for protons which shields the redox reaction sites at the outer side of the membrane against the outer aqueous phase. This barrier can be lowered by sand grinding the chloroplasts, by digitonin treatment and by uncoupling agents. At the extreme the barrier can be practically eliminated to yield rates of proton uptake matching the rates of the corresponding redox reactions. This gives conclusive evidence that the electrochemical potential difference which light induces across the functional membrane of photosynthesis is generated by a vectorial electron-hydrogen transport system as postulated by Mitchell (e.g. (1966) Biol. Rev. 41, 445–502).     

Quantitation of physical-chemical properties of the aqueous phase inside the phoE ionic channel.

The anion-specific channel of the phoE porine is a miniature body of water surrounded by peptide walls. The physical and chemical properties of the water in such a microscopic space were measured by monitoring the dynamics of a well-studied reaction--the protolytic dissociation of a strong acid. To attain this purpose, we allowed pyranine (8-hydroxypyrene-1,3,6-trisulfonate) to bind to the anion-specific channel. The dye is bound, with a 1:1 stoichiometry, with a delta G = -9.5 kcal/mol. Photoexcitation of the dye, to its first electronic singlet state (phi OH*), renders it very acidic and the hydroxyl proton dissociates to H+ and excited anion (phi O*-). We employed single photon-counting time-resolved fluorimetry, to monitor the reversible dissociation of pyranine as it proceeds within the channel and reconstructed the observed signal by a numerical integration of the differential diffusion equation pertinent for a proton within the channel. The most characteristic feature of the water-filled channel, is the intensified electrostatic interactions attained by the low dielectric constant of the diffusion space, epsilon eff = 24. For this reason, the electric field of a few positive charges is sufficient to ensure that an anion entering the channel will be effectively sucked in. The interaction of the water molecules with the peptide structure forming the channel affects the physical properties of the water. Their capacity to conduct proton, quantitated by the protons diffusion coefficient (4.5.10(-5) cm2/s), is reduced by 50% with respect to that of bulk water. The activity of the water in the channel is reduced to alpha H2O = 0.966. These observation are in accord with our previous studies of water in small defined cavities in proteins.     

Proton-collecting properties of bovine heart cytochrome C oxidase: kinetic and electrostatic analysis.

Proton-transfer reactions on the surface of bovine heart cytochrome c oxidase were investigated by combining a laser-induced proton-pulse technique with molecular modeling. The experimental approach simultaneously monitors the state of pyranine protonation in the bulk phase and that of a fluorescein indicator specifically attached to the native Cys(III-115) residue of subunit III of cytochrome oxidase. The reversible dynamics of the acid-base equilibration between the surface and the bulk phase were measured with submicrosecond time resolution and analyzed by numerical integration of coupled nonlinear differential rate equations. Kinetic analysis shows that carboxylates on the surface of the protein act as a proton-collecting antenna, which is able to rapidly transfer protons to nearby histidines that function as a local proton reservoir. These properties enable cytochrome oxidase to carry out its redox-linked proton translocation. Molecular modeling of the fluorescein-binding site indicates that, in addition to the covalent bond, the dye is anchored through a hydrogen bond to the hydroxyl moiety of Tyr(VII-50). The protonation of the dye is mediated through three residues that shuttle protons between the bulk and the dye. A correlation between the measured kinetic properties of the bound fluorescein and the different configurations of the dye allows us to predict the identity of the proton-binding sites in the fluorescein-binding domain.     

Probing biological interfaces by tracing proton passage across them.

The properties of water at the surface, especially at an electrically charged one, differ essentially from those in the bulk phase. Here we survey the traits of surface water as inferred from proton pulse experiments with membrane enzymes. In such experiments, protons that are ejected (or captured) by light-triggered enzymes are traced on their way between the membrane surface and the bulk aqueous phase. In several laboratories it has been shown that proton exchange between the membrane surface and the bulk aqueous phase takes as much as about 1 ms, but could be accelerated by added mobile pH-buffers. Since the accelerating capacity of the latter decreased with increase in their electric charge, it was suggested that the membrane surface is separated from the bulk aqueous phase by a barrier of electrostatic nature. In terms of ordinary electrostatics, the barrier could be ascribed to dielectric saturation of water at a charged surface. In terms of nonlocal electrostatics, the barrier could result from the dielectric overscreening in the surface water layers. It is discussed how the interfacial potential barrier can affect the reactions at interface, especially those coupled with biological energy conversion and membrane transport.     

Far-field analysis of coupled bulk and boundary layer diffusion toward an ion channel entrance.

We present a far-field analysis of ion diffusion toward a channel embedded in a membrane with a fixed charge density. The Smoluchowski equation, which represents the 3D problem, is approximated by a system of coupled three- and two-dimensional diffusions. The 2D diffusion models the quasi-two-dimensional diffusion of ions in a boundary layer in which the electrical potential interaction with the membrane surface charge is important. The 3D diffusion models ion transport in the bulk region outside the boundary layer. Analytical expressions for concentration and flux are developed that are accurate far from the channel entrance. These provide boundary conditions for a numerical solution of the problem. Our results are used to calculate far-field ion flows corresponding to experiments of Bell and Miller (Biophys. J. 45:279, 1984).     

Proton transfer dynamics on the surface of the late M state of bacteriorhodopsin

The cytoplasmic surface of the BR (initial) state of bacteriorhodopsin is characterized by a cluster of three carboxylates that function as a proton-collecting antenna. Systematic replacement of most of the surface carboxylates indicated that the cluster is made of D104, E161, and E234 (Checover, S., Y. Marantz, E. Nachliel, M. Gutman, M. Pfeiffer, J. Tittor, D. Oesterhelt, and N. Dencher. 2001. Biochemistry. 40:4281-4292), yet the BR state is a resting configuration; thus, its proton-collecting antenna can only indicate the presence of its role in the photo-intermediates where the protein is re-protonated by protons coming from the cytoplasmic matrix. In the present study we used the D96N and the triple (D96G/F171C/F219L) mutant for monitoring the proton-collecting properties of the protein in its late M state. The protein was maintained in a steady M state by continuous illumination and subjected to reversible pulse protonation caused by repeated excitation of pyranine present in the reaction mixture. The re-protonation dynamics of the pyranine anion was subjected to kinetic analysis, and the rate constants of the reaction of free protons with the surface groups and the proton exchange reactions between them were calculated. The reconstruction of the experimental signal indicated that the late M state of bacteriorhodopsin exhibits an efficient mechanism of proton delivery to the unoccupied-most basic-residue on its cytoplasmic surface (D38), which exceeds that of the BR configuration of the protein. The kinetic analysis was carried out in conjunction with the published structure of the M state (Sass, H., G. Büldt, R. Gessenich, D. Hehn, D. Neff, R. Schlesinger, J. Berendzen, and P. Ormos. 2000. Nature. 406:649-653), the model that resolves most of the cytoplasmic surface. The combination of the kinetic analysis and the structural information led to identification of two proton-conducting tracks on the protein's surface that are funneling protons to D38. One track is made of the carboxylate moieties of residues D36 and E237, while the other is made of D102 and E232. In the late M state the carboxylates of both tracks are closer to D38 than in the BR (initial) state, accounting for a more efficient proton equilibration between the bulk and the protein's proton entrance channel. The triple mutant resembles in the kinetic properties of its proton conducting surface more the BR-M state than the initial state confirming structural similarities with the BR-M state and differences to the BR initial state.     

Probing of the substrate binding domain of lactose permease by a proton pulse

The lactose permease of Escherichia coli coupled proton transfer across the bacterial inner membrane with the uptake of beta-galactosides. In the present study we have used the cysteine-less C148 mutant that was selectively labeled by fluorescein maleimide on the C148 residue, which is an active component of the substrate transporting cavity. Measurements of the protonation dynamics of the bound pH indicator in the time resolved domain allowed us to probe the binding site by a free diffusing proton. The measured signal was reconstructed by numeric integration of differential rate equations that comply with the detailed balance principle and account for all proton transfer reactions taking place in the reaction mixture. This analysis yields the rate constants and pK values of all residues participating in the fast proton transfer reaction between the bulk and the protein's surface, revealing the exposed residues that react with free protons in a diffusion controlled reaction and how they transfer protons among themselves. The magnitudes of these rate constants were finally evaluated by comparison with the rate predicted by the Debye-Smoluchowski equation. The analysis of the kinetic and pK values indicated that the protein-fluorescein adduct assumes two conformation states. One is dominant above pH 7.4, while the other exists only below 7.1. In the high pH range, the enzyme assumes a constrained configuration and the rate constant of the reaction of a free diffusing proton with the bound dye is 10 times slower than a diffusion controlled reaction. In this state, the carboxylate moiety of residue E126 is in close proximity to the dye and exchanges a proton with it at a very fast rate. Below pH 7.1, the substrate binding domain is in a relaxed configuration and freely accessed by bulk protons, and the rate of proton exchange between the dye and E126 is 100,000 times slower. The relevance of these observations to the catalytic cycle is discussed.     

Protons @ interfaces: implications for biological energy conversion

The review focuses on the anisotropy of proton transfer at the surface of biological membranes. We consider (i) the data from "pulsed" experiments, where light-triggered enzymes capture or eject protons at the membrane surface, (ii) the electrostatic properties of water at charged interfaces, and (iii) the specific structural attributes of proton-translocating enzymes. The pulsed experiments revealed that proton exchange between the membrane surface and the bulk aqueous phase takes as much as about 1 ms, but could be accelerated by added mobile pH-buffers. Since the accelerating capacity of the latter decreased with the increase in their electric charge, it was concluded that the membrane surface is separated from the bulk aqueous phase by a barrier of electrostatic nature. The barrier could arise owing to the water polarization at the negatively charged membrane surface. The barrier height depends linearly on the charge of penetrating ions; for protons, it has been estimated as about 0.12 eV. While the proton exchange between the surface and the bulk aqueous phase is retarded by the interfacial barrier, the proton diffusion along the membrane, between neighboring enzymes, takes only microseconds. The proton spreading over the membrane is facilitated by the hydrogen-bonded networks at the surface. The membrane-buried layers of these networks can eventually serve as a storage/buffer for protons (proton sponges). As the proton equilibration between the surface and the bulk aqueous phase is slower than the lateral proton diffusion between the "sources" and "sinks", the proton activity at the membrane surface, as sensed by the energy transducing enzymes at steady state, might deviate from that measured in the adjoining water phase. This trait should increase the driving force for ATP synthesis, especially in the case of alkaliphilic bacteria.     

Kinetic analysis of protonation of a specific site on a buffered surface of a macromolecular body

The kinetics of protonation of a specific site on a macromolecular structure (micelle) in buffered solution was studied with the purpose of evaluating the effect of buffer on the observed dynamics. The experimental system consisted of the following elements: Brij 58 micelles serving as homogeneous uncharged macromolecular bodies, bromocresol green, a well-adsorbed proton detector, and 2-naphthol-3,6-disulfonate as a proton emitter in the bulk. Imidazole was the mobile buffer while neutral red, which has a high affinity for the micellar surface, served as the immobile buffer. An intensive laser pulse ejects a proton from the proton emitter, and the subsequent proton-transfer reactions are measured by fast spectrophotometric methods. The dynamics of proton pulse in buffered solution are characterized by a very rapid trapping of the discharged protons by the abundant buffer molecules. This event has a major effect on the kinetic regime of the reaction. During the first 200 ns the proton flux is rate limited by free-proton diffusion. After this period, when the free-proton concentration decayed to the equilibrium level, the relaxation of the system is carried out by the diffusion of buffer. Thus in the buffered biochemical system, at neutral pH, most of proton flux between active sites and bulk is carried out by buffer molecules--not by diffusion of free protons. Surface groups on a high molecular weight body exchange protons among them at a very fast rate. This reaction has a major role on proton transfer from a specific site to the bulk.(ABSTRACT TRUNCATED AT 250 WORDS)     

Energization-induced redistribution of charge carriers near membranes

The electric field arising from proton pumping across a topologically closed biological membrane causes accumulation close to the membrane of ionic charges equivalent to the charge of the pumped protons, positive on the side towards which protons are pumped, negative on the other side. We shall call this the 'active surface charge'. We here use the Poisson-Boltzmann equation to evaluate the effects of zwitterionic buffer molecules and uncharged proteins in the aqueous phase bordering the membrane on the magnitude and ionic composition of the active surface charge. For the positive side of the membrane, the main results are: (1) If the membrane is freely accessible to bulk phase ions, pumped protons exchange with these ions, such that the active surface charge consists of salt cations. (2) If a significant fraction of the ions in bulk solution consists of buffer molecules, then some of the pumped protons will remain close to the membrane and constitute a major fraction of the active surface charge. (3) If a protein layer borders the membrane, a significant part of the transmembrane electric potential difference exists within that protein layer and protons inside this layer dominate the active surface charge. (4) On the negative side of the membrane the corresponding phenomena would occur. (5) All these effects are strictly dependent on the transmembrane electric potential difference arising from proton pumping and would come in addition to the well known effects of buffers and electrically charged proteins on the retention of scalar protons. (6) No additional proton diffusion barrier may be required to account for a deficit in number of protons observed in the aqueous bulk phase upon aeration-induced proton pumping.     

Protons @ interfaces: Implications for biological energy conversion

The review focuses on the anisotropy of proton transfer at the surface of biological membranes. We consider (i) the data from “pulsed” experiments, where light-triggered enzymes capture or eject protons at the membrane surface, (ii) the electrostatic properties of water at charged interfaces, and (iii) the specific structural attributes of proton-translocating enzymes. The pulsed experiments revealed that proton exchange between the membrane surface and the bulk aqueous phase takes as much as about 1 ms, but could be accelerated by added mobile pH-buffers. Since the accelerating capacity of the latter decreased with the increase in their electric charge, it was concluded that the membrane surface is separated from the bulk aqueous phase by a barrier of electrostatic nature. The barrier could arise owing to the water polarization at the negatively charged membrane surface. The barrier height depends linearly on the charge of penetrating ions; for protons, it has been estimated as about 0.12 eV. While the proton exchange between the surface and the bulk aqueous phase is retarded by the interfacial barrier, the proton diffusion along the membrane, between neighboring enzymes, takes only microseconds. The proton spreading over the membrane is facilitated by the hydrogen-bonded networks at the surface. The membrane-buried layers of these networks can eventually serve as a storage/buffer for protons (proton sponges). As the proton equilibration between the surface and the bulk aqueous phase is slower than the lateral proton diffusion between the “sources” and “sinks”, the proton activity at the membrane surface, as sensed by the energy transducing enzymes at steady state, might deviate from that measured in the adjoining water phase. This trait should increase the driving force for ATP synthesis, especially in the case of alkaliphilic bacteria.     

Mechanisms of generation of local ΔpH in mitochondria and bacteria

The concepts of global and local coupling between proton generators, the enzymes of the respiratory chain, and the consumer, the ATP synthase, coexist in the theory of oxidative phosphorylation. Global coupling is trivial proton transport via the aqueous medium, whereas local coupling implies that the protons pumped are consumed before they escape to the bulk phase. In this work, the conditions for the occurrence of local coupling are explored. It is supposed that the membrane retains protons near its surface and that the proton current generated by the proton pumps rapidly decreases with increasing proton motive force (pmf). It is shown that the competition between the processes of proton translocation across the membrane and their dissipation from the surface to the bulk can result in transient generation of a local ΔpH in reply to a sharp change in pmf; the appearance of local ΔpH, in turn, leads to rapid recovery of the pmf, and hence, it provides for stabilization of the potential at the membrane. Two mechanisms of such kind are discussed: 1) pH changes in the surface area due to proton pumping develop faster than those due to proton escape to the bulk; 2) the former does not take place, but the protons leaving the surface do not equilibrate with the bulk immediately; rather, they give rise to a non-equilibrium concentration near the surface and, as a result, to a back proton flow to the surface. The first mechanism is more efficient, but it does not occur in mitochondria and neutrophilic bacteria, whereas the second can produce ΔpH on the order of unity. In the absence of proton retardation at the surface, local ΔpH does not arise, whereas the formation of global ΔpH is possible only at buffer concentration of less than 10 mM. The role of the mechanisms proposed in transitions between States 3 and 4 of the respiratory chain is discussed. The main conclusion is that surface protons, under conditions where they play a role, support stabilization of the membrane pmf and rapid communication between proton generators and consumers, while their contribution to the energetics is not significant.     

Kinetics of proton diffusion in the regimes of fast and slow exchange between the membrane surface and the bulk solution

The phenomenological model developed in our recent publications [9,10] is used to investigate the kinetics of proton diffusion from a source to a detector on the membrane surface. In most cases the observed kinetics shows a single diffusional maximum with the exponential ascending front and the power-law descending tail. The kinetics depends on the distance between the source and the detector. If the detector is located inside the proton collecting antenna, the kinetics corresponds to the surface diffusion at the times near the maximum and shortly thereafter, and it turns into the bulk diffusion kinetics at longer times, after the equilibrium is established between the membrane surface and the bulk solution. If the detector is located outside the antenna, the kinetics corresponds to the bulk diffusion at all times where the signal is nonvanishing. What is seen at locations near the antenna radius depends on the exchange regime. In the regime of fast exchange between the surface and the bulk as compared to the bulk diffusion, the kinetics shows a single peak whose location is intermediate between the peaks for the surface and bulk diffusion. In the regime of slow exchange there are two maxima corresponding to the surface and bulk diffusion. In buffered solutions the antenna radius decreases with increasing buffer concentration, which changes the kinetics from the surface to bulk diffusion. The theory is applied to interprete recent experiments on a phospholipid membrane [25]. It is found that (i) the fast exchange regime is operating since only a single maximum is observed; (ii) the shift of the maximum toward longer times with increasing buffer concentration is a manifestation of the transition from the surface to bulk diffusion kinetics. The authors are grateful to Yu. N. Antonenko and A. I. Kotelnikov for helpful discussions. This work was supported by the Russian Foundation for Basic Research (05-03-32104), U.S. Civilian Research and Development Foundation (RUC2-2658-MO-05), U. S. National Science Foundation, and U. S. National Institutes of Health.     

Kinetic analysis of proton transfer between reactants adsorbed to the same micelle. The effect of proximity on the rate constants

The dense packing of protogenic enzymes on the coupling membrane can furnish a route for a rapid proton flux which may avoid the adjacent bulk phase. In order to evaluate the role of proximity between reactants on the rate constant of proton transfer we generated a model system consisting of 2-naphthol and pH indicator (bromocresol green) both adsorbed on the same micelle of unchanged detergent. Excitation of the 2-naphthol by a short intensive laser pulse lowers its pK with subsequent synchronized proton ejection. The discharged protons are detected by their reaction with the indicator using a fast transient absorption technique. Evidence is produced that under certain conditions all of the observed proton flux represents proton transfer between 2-naphthol and indicator molecules sharing the same micelle. In this model system the entire proton flux proceeds through an aqueous phase fully accessible to phosphate ions. The high proximity of the reactants (the separation can not exceed approximately 6 nm) has a marked effect on the rate constant of the reaction k = 2.0 +/- 0.5 X 10(11) M-1 s-1. In spite of this extremely fast rate of reaction we observe unhindered competition, for the surface discharged proton, between the surface-bound reactants and phosphate ions in the bulk. Thus even in proton transfer between closely packed reactants on an interface, the diffusion of the proton is not limited to the interface. This finding implies that on bioenergetic surface the electrochemical potential of the proton on the surface will equal that of the bulk.     

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).     

An integrated field-effect microdevice for monitoring membrane transport in Xenopus laevis oocytes via lateral proton diffusion

An integrated microdevice for measuring proton-dependent membrane activity at the surface of Xenopus laevis oocytes is presented. By establishing a stable contact between the oocyte vitelline membrane and an ion-sensitive field-effect (ISFET) sensor inside a microperfusion channel, changes in surface pH that are hypothesized to result from facilitated proton lateral diffusion along the membrane were detected. The solute diffusion barrier created between the sensor and the active membrane area allowed detection of surface proton concentration free from interference of solutes in bulk solution. The proposed sensor mechanism was verified by heterologously expressing membrane transport proteins and recording changes in surface pH during application of the specific substrates. Experiments conducted on two families of phosphate-sodium cotransporters (SLC20 & SLC34) demonstrated that it is possible to detect phosphate transport for both electrogenic and electroneutral isoforms and distinguish between transport of different phosphate species. Furthermore, the transport activity of the proton/amino acid cotransporter PAT1 assayed using conventional whole cell electrophysiology correlated well with changes in surface pH, confirming the ability of the system to detect activity proportional to expression level.     

Conductance routes for protons across membrane barriers

Simple phospholipid bilayers show a high level of permeability to protons; in spite of this fact, large proton gradients existing across such bilayers may decay very slowly. In sealed systems, the free movement of protons across a membrane barrier is severely restricted by the coincident development of a proton diffusion potential. Using the fluorescent weak acid N-[5-(dimethylamino)naphth-1-ylsulfonyl]glycine strongly buffered systems movement of the small number of protons giving rise to this electrical potential is insufficient to perturb the proton concentration gradient; significant flux of protons (and hence significant collapse of the concentration gradient) can only occur if protons traverse the membrane as part of an electroneutral complex or if there is a balancing flow of appropriate counterions. In both instances, proton flux is obligatorily coupled to the translocation of species other than protons. In weakly buffered systems, the small initial uncoupled electrogenic flux of protons may significantly alter the concentration gradient. This initial rapid gradient collapse caused by uncoupled electrogenic proton movements is then superimposed upon the residual collapse attributable to tightly coupled proton flux. The initial uncoupled electrogenic proton flux shows a temperature dependence very similar to that demonstrated for water permeation across simple lipid bilayers; upon cooling, there is a sharp decrease in flux at the temperature coinciding with the main gel-liquid-crystalline phase transition of the lipid. The coupled proton flux shows a markedly different temperature dependence with no dramatic change in rate at the phase transition temperature and strong similarity to the behavior previously seen with solutes known to be permeating as electrically neutral compounds.(ABSTRACT TRUNCATED AT 250 WORDS)     

G204D, a mutation that blocks the proton-conducting D-channel of the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides

Cytochrome c oxidase uses the free energy of oxygen reduction to establish a transmembrane proton gradient. The proton-conducting D-channel in this enzyme is the major input pathway for protons which go to the binuclear center for water formation ("chemical protons") and likely the only input pathway for protons that get translocated across the lipid membrane ("pumped protons"). The D-channel starts at an acidic residue near the protein surface (D132, Rhodobacter sphaeroides numbering) and leads to another acidic residue near the binuclear center. Recent studies have shown that mutants that introduce an additional acidic residue in the channel (N139D) have the remarkable effect of accelerating steady-state oxidase activity but completely eliminating proton pumping. In this work, an aspartic acid was introduced at the position of glycine 204, G204D, which is also within the D-channel, and the effects were examined. In contrast to N139D, the G204D mutation results in a dramatic decrease of the steady-state oxygen reductase activity (<2% of wild type) [Aagaard, A., and Brzezinski, P. (2001) FEBS Lett. 494, 157-160]. The residual activity is not coupled to the proton pump, and furthermore, in reconstituted vesicles the mutant enzyme exhibits a reverse respiration control ratio; i.e., the mutant oxidase activity is stimulated rather than inhibited when working against a protonmotive force. Hence, the mutant behaves very much like the D132N, which blocks proton uptake through the D-channel. Single-turnover experiments show that the rate-limiting step in the reaction of O2 with the fully reduced G204D mutant is the F --> O transition, similar to the D132N mutant. The block of the D-channel in the D132N mutant can be partly bypassed by biochemically removing subunit III from the enzyme, indicating that removal of the subunit reveals an alternate entrance for protons to the channel. However, this is not observed with the G204D mutant. This suggests that the cryptic entrance to the D-channel that is revealed by the removal of subunit III is between the levels of G204 and D132.