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Peter Brzezinski

Publications and source records attributed to Peter Brzezinski.

At least 19 recordsLinked to original sources
PubMed2006

Localized proton microcircuits at the biological membrane-water interface.

Cellular processes such as nerve conduction, energy metabolism, and import of nutrients into cells all depend on transport of ions across biological membranes through specialized membrane-spanning proteins. Understanding these processes at a molecular level requires mechanistic insights into the interaction between these proteins and the membrane itself. To explore the role of the membrane in ion translocation we used an approach based on fluorescence correlation spectroscopy. Specifically, we investigated exchange of protons between the water phase and the membrane surface, as well as diffusion of protons along membrane surfaces, at a single-molecule level. We show that the lipid head groups collectively act as a proton-collecting antenna, dramatically accelerating proton uptake from water to a membrane-anchored proton acceptor. Furthermore, the results show that proton transfer along the surface can be significantly faster than that between the lipid head groups and the surrounding water phase. Thus, ion translocation across membranes and between the different membrane protein components is a complex interplay between the proteins and the membrane itself, where the membrane acts as a proton-conducting link between membrane-spanning proton transporters.

Kinetics↗
PubMed2006

Replacing Asn207 by aspartate at the neck of the D channel in the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides results in decoupling the proton pump.

Cytochrome oxidase catalyzes the reduction of O2 to water and conserves the considerable free energy available from this reaction in the form of a proton motive force. For each electron, one proton is electrogenically pumped across the membrane. Of particular interest is the mechanism by which the proton pump operates. Previous studies of the oxidase from Rhodobacter sphaeroides have shown that all of the pumped protons enter the enzyme through the D channel and that a point mutant, N139D, in the D channel completely eliminates proton pumping without reducing oxidase activity. N139 is one of three asparagines near the entrance of the D channel, where there is a narrowing or neck, through which a single file of water molecules pass. In the current work, it is shown that replacement of a second asparagine in this region by an asparate, N207D, also decouples the proton pump without altering the oxidase activity of the enzyme. Previous studies demonstrated that the N139D mutant results in an increase in the apparent pKa of E286, a functionally critical residue that is located 20 A away from N139 at the opposite end of the D channel. In the current work, it is shown that the N207 mutation also increases the apparent pKa of E286. This finding reinforces the proposal that the elimination of proton pumping is the result of an increase of the apparent proton affinity of E286, which, in turn, prevents the timely proton transfer to a proton accepter group within the exit channel of the proton pump.

Asparagine↗
PubMed2006

Mapping protein dynamics in catalytic intermediates of the redox-driven proton pump cytochrome c oxidase.

Redox-driven proton pumps such as cytochrome c oxidase (CcO) are fundamental elements of the energy transduction machinery in biological systems. CcO is an integral membrane protein that acts as the terminal electron acceptor in respiratory chains of aerobic organisms, catalyzing the four-electron reduction of O2 to H2O. This reduction also requires four protons taken from the cytosolic or negative side of the membrane, with an additional uptake of four protons that are pumped across the membrane. Therefore, the proton pump must embody a "gate," which provides alternating access of protons to one or the other side of the membrane but never both sides simultaneously. However, the exact mechanism of proton translocation through CcO remains unknown at the molecular level. Understanding pump function requires knowledge of the nature and location of these structural changes that is often difficult to access with crystallography or NMR spectroscopy. In this paper, we demonstrate, with amide hydrogen/deuterium exchange MS, that transitions between catalytic intermediates in CcO are orchestrated with opening and closing of specific proton pathways, providing an alternating access for protons to the two sides of the membrane. An analysis of these results in the framework of the 3D structure of CcO indicate the spatial location of a gate, which controls the unidirectional proton flux through the enzyme and points to a mechanism by which CcO energetically couples electron transfer to proton translocation.

Biological Transport↗
PubMed2006

Design principles of proton-pumping haem-copper oxidases.

Transmembrane electrochemical proton gradients are used to store free energy in biological systems, and to drive the synthesis of biomolecules and transmembrane transport. These gradients are maintained by membrane-bound proton transporters that employ free energy provided by, for example, electron transfer or light. In recent years, the structures of several membrane proteins involved in proton translocation have been determined, and indicate that both protein-bound water molecules and protonatable amino acid residues play central roles in transmembrane proton conduction. From these structures, in combination with functional studies, have emerged general principles of proton transfer across membranes and control mechanisms for such reactions, in particular with regard to the electron-transfer-driven proton pump cytochrome c oxidase.

Animals↗
PubMed2006

Real-time transmembrane translocation of penetratin driven by light-generated proton pumping.

Cell penetrating peptides (CPPs) are small peptides that are able to penetrate the plasma membrane of mammalian cells. Because these peptides can also carry large hydrophilic cargos such as proteins, they could potentially be used to transport biologically active drugs across cell membranes to modulate in vivo biology. One characteristic feature of the CPPs is that they typically have a net positive charge. Therefore, a key issue associated with the transport mechanism is the role of the transmembrane electrochemical potential in driving the peptides across the membrane. In this study, we have reconstituted bacteriorhodopsin (bR) in large unilamellar vesicles (LUVs) with fluorescein-labeled CPP penetratin enclosed within the LUVs under conditions when the fluorescence is quenched. Illumination of the bacteriorhodopsin-containing LUVs resulted in creation of a transmembrane proton electrochemical gradient (positive on the inside). Upon generation of this gradient, an increase in fluorescence was observed, which shows that the proton gradient drives the translocation of penetratin. The mechanism most likely can be generalized to other CPPs.

Bacteriorhodopsins↗
PubMed2006

Transmembrane proton translocation by cytochrome c oxidase.

Respiratory heme-copper oxidases are integral membrane proteins that catalyze the reduction of molecular oxygen to water using electrons donated by either quinol (quinol oxidases) or cytochrome c (cytochrome c oxidases, CcOs). Even though the X-ray crystal structures of several heme-copper oxidases and results from functional studies have provided significant insights into the mechanisms of O2 -reduction and, electron and proton transfer, the design of the proton-pumping machinery is not known. Here, we summarize the current knowledge on the identity of the structural elements involved in proton transfer in CcO. Furthermore, we discuss the order and timing of electron-transfer reactions in CcO during O2 reduction and how these reactions might be energetically coupled to proton pumping across the membrane.

Biological Transport↗
PubMed2006

Inhibition of proton pumping by zinc ions during specific reaction steps in cytochrome c oxidase.

Cytochrome c oxidase (CytcO) is a redox-driven proton pump in the respiratory chain of mitochondria and many aerobic bacteria. The results from several studies have shown that zinc ions interfere with both the uptake and release of protons, presumably by binding near the orifice of the proton entrance and exit pathways. To elucidate the effect of Zn2+ binding on individual electron and proton-transfer reactions, in this study, we have investigated the reaction of the fully reduced R. sphaeroides CytcO with O2, both with enzyme in detergent solution and reconstituted in phospholipid vesicles, and, with and without, Zn2+. The results show that addition of Zn2+ at concentrations of < or = 250 microM to the outside of the vesicles did not alter the transition rates between intermediates PR (P3)-->F3-->O4. However, proton pumping was impaired specifically during the P3-->F3, but not during the F3-->O4 transition at Zn2+ concentrations of < or = 25 microM. Furthermore, proton pumping during the P3-->F3 transition was typically impaired with the "as isolated" CytcO, which was found to contain Zn2+ ions at microM concentration. As has already been shown, Zn2+ was also found to obstruct proton uptake during the P3-->F3 transition, presumably by binding to a site near the orifice of the D-pathway. In this work we found a KI of approximately 1 microM for this binding site. In conclusion, the results show that Zn2+ ions bind on both sides of CytcO and that binding of Zn2+ at the proton output side selectively impairs proton release during the P3-->F3 transition.

Biological Transport↗
PubMed2006

Controlled uncoupling and recoupling of proton pumping in cytochrome c oxidase.

Cytochrome c oxidase (CcO) is the terminal enzyme of the respiratory chain and couples energetically the reduction of oxygen to water to proton pumping across the membrane. The results from previous studies showed that proton pumping can be uncoupled from the O2-reduction reaction by replacement of one single residue, Asn-139 by Asp (N139D), located approximately 30 A from the catalytic site, in the D-proton pathway. The uncoupling was correlated with an increase in the pK(a) of an internal proton donor, Glu-286, from approximately 9.4 to >11. Here, we show that replacement of the acidic residue, Asp-132 by Asn in the N139D CcO (D132N/N139D double-mutant CcO) results in restoration of the Glu-286 pK(a) to the original value and recoupling of the proton pump during steady-state turnover. Furthermore, a kinetic investigation of the specific reaction steps in the D132N/N139D double-mutant CcO showed that proton pumping is sustained even if proton uptake from solution, through the D-pathway, is slowed. However, during single-turnover oxidation of the fully reduced CcO the P --> F transition, which does not involve electron transfer to the catalytic site, was not coupled to proton pumping. The results provide insights into the mechanism of proton pumping by CcO and the structural elements involved in this process.

Catalysis↗
PubMed2005

The timing of proton migration in membrane-reconstituted cytochrome c oxidase.

In mitochondria and aerobic bacteria energy conservation involves electron transfer through a number of membrane-bound protein complexes to O2. The reduction of O2, accompanied by the uptake of substrate protons to form H2O, is catalyzed by cytochrome c oxidase (CcO). This reaction is coupled to proton translocation (pumping) across the membrane such that each electron transfer to the catalytic site is linked to the uptake of two protons from one side and the release of one proton to the other side of the membrane. To address the mechanism of vectorial proton translocation, in this study we have investigated the solvent deuterium isotope effect of proton-transfer rates in CcO oriented in small unilamellar vesicles. Although in H2O the uptake and release reactions occur with the same rates, in D2O the substrate and pumped protons are taken up first (tau(D) congruent with 200 micros, "peroxy" to "ferryl" transition) followed by a significantly slower proton release to the other side of the membrane (tau(D) congruent with 1 ms). Thus, the results define the order and timing of the proton transfers during a pumping cycle. Furthermore, the results indicate that during CcO turnover internal electron transfer to the catalytic site is controlled by the release of the pumped proton, which suggests a mechanism by which CcO orchestrates a tight coupling between electron transfer and proton translocation.

Detergents↗
PubMed2005

A mechanistic principle for proton pumping by cytochrome c oxidase.

In aerobic organisms, cellular respiration involves electron transfer to oxygen through a series of membrane-bound protein complexes. The process maintains a transmembrane electrochemical proton gradient that is used, for example, in the synthesis of ATP. In mitochondria and many bacteria, the last enzyme complex in the electron transfer chain is cytochrome c oxidase (CytcO), which catalyses the four-electron reduction of O2 to H2O using electrons delivered by a water-soluble donor, cytochrome c. The electron transfer through CytcO, accompanied by proton uptake to form H2O drives the physical movement (pumping) of four protons across the membrane per reduced O2. So far, the molecular mechanism of such proton pumping driven by electron transfer has not been determined in any biological system. Here we show that proton pumping in CytcO is mechanistically coupled to proton transfer to O2 at the catalytic site, rather than to internal electron transfer. This scenario suggests a principle by which redox-driven proton pumps might operate and puts considerable constraints on possible molecular mechanisms by which CytcO translocates protons.

Binding Sites↗
PubMed2005

The protonation state of a heme propionate controls electron transfer in cytochrome c oxidase.

In cytochrome c oxidase (CcO), exergonic electron transfer reactions from cytochrome c to oxygen drive proton pumping across the membrane. Elucidation of the proton pumping mechanism requires identification of the molecular components involved in the proton transfer reactions and investigation of the coupling between internal electron and proton transfer reactions in CcO. While the proton-input trajectory in CcO is relatively well characterized, the components of the output pathway have not been identified in detail. In this study, we have investigated the pH dependence of electron transfer reactions that are linked to proton translocation in a structural variant of CcO in which Arg481, which interacts with the heme D-ring propionates in a proposed proton output pathway, was replaced with Lys (RK481 CcO). The results show that in RK481 CcO the midpoint potentials of hemes a and a(3) were lowered by approximately 40 and approximately 15 mV, respectively, which stabilizes the reduced state of Cu(A) during reaction of the reduced CcO with O(2). In addition, while the pH dependence of the F --> O rate in wild-type CcO is determined by the protonation state of two protonatable groups with pK(a) values of 6.3 and 9.4, only the high-pK(a) group influences this rate in RK481 CcO. The results indicate that the protonation state of the Arg481 heme a(3) D-ring propionate cluster having a pK(a) of approximately 6.3 modulates the rate of internal electron transfer and may act as an acceptor of pumped protons.

Animals↗
PubMed2004

A single-amino-acid lid renders a gas-tight compartment within a membrane-bound transporter.

Proteins undergo structural fluctuations between nearly isoenergetic substates. Such fluctuations are often intimately linked with the functional properties of proteins. However, in some cases, such as in transmembrane ion transporters, the control of the ion transport requires that the protein is designed to restrict the motions in specific regions. In this study, we have investigated the dynamics of a membrane-bound respiratory oxidase, which acts both as an enzyme catalyzing reduction of O(2) to H(2)O and as a transmembrane proton pump. The segment of the protein where proton translocation is controlled ("gating" region) overlaps with a channel through which O(2) is delivered to the catalytic site. We show that the replacement of an amino acid residue with a small side chain (Gly) by one with a larger side chain (Val), in a narrow part of this channel, completely blocks the O(2) access to the catalytic site and results in formation of a compartment around the site that is impermeable to small gas molecules. Thus, the protein motions cannot counter the blockage introduced by the mutation. These results indicate that the protein motions are restricted in the proton-gating region and that rapid O(2) delivery to the catalytic site requires a gas channel, which is confined within a rigid protein body.

Amino Acids↗
PubMed2004

Development of a bacterial biosensor for nitrotoluenes: the crystal structure of the transcriptional regulator DntR.

The transcriptional regulator DntR, a member of the LysR family, is a central element in a prototype bacterial cell-based biosensor for the detection of hazardous contamination of soil and groundwater by dinitrotoluenes. To optimise the sensitivity of the biosensor for such compounds we have chosen a rational design of the inducer-binding cavity based on knowledge of the three-dimensional structure of DntR. We report two crystal structures of DntR with acetate (resolution 2.6 angstroms) and thiocyanate (resolution 2.3 angstroms), respectively, occupying the inducer-binding cavity. These structures allow for the construction of models of DntR in complex with salicylate (Kd approximately or = 4 microM) and 2,4-dinitrotoluene that provide a basis for the design of mutant DntR with enhanced specificity for dinitrotoluenes. In both crystal structures DntR crystallises as a homodimer with a "head-to-tail" arrangement of monomers in the asymmetric unit. Analysis of the crystal structure has allowed the building of a full-length model of DntR in its biologically active homotetrameric form consisting of two "head-to-head" dimers. The implications of this model for the mechanism of transcription regulation by LysR proteins are discussed.

Base Sequence↗
PubMed2004

Structural elements involved in electron-coupled proton transfer in cytochrome c oxidase.

Haem-copper oxidases are the last components of the respiratory chains in aerobic organisms. These membrane-bound enzymes energetically couple the electron transfer (eT) reactions associated with reduction of dioxygen to water, to proton pumping across the membrane. Even though the mechanism of proton pumping at the molecular level still remains to be uncovered, recent progress has presented us with the structural features of the pumping machinery and detailed information about the eT and proton-transfer reactions associated with the pumping process.

Bacterial Proteins↗
PubMed2004

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).

Electron Transport↗
PubMed2003

Redox-coupled proton translocation in biological systems: proton shuttling in cytochrome c oxidase.

In the respiratory chain free energy is conserved by linking the chemical reduction of dioxygen to the electrogenic translocation of protons across a membrane. Cytochrome c oxidase (CcO) is one of the sites where this linkage occurs. Although intensively studied, the molecular mechanism of proton pumping by this enzyme remains unknown. Here, we present data from an investigation of a mutant CcO from Rhodobacter sphaeroides [Asn-139 --> Asp, ND(I-139)] in which proton pumping is completely uncoupled from the catalytic turnover (i.e., reduction of O2). However, in this mutant CcO, the rate by which O2 is reduced to H2O is even slightly higher than that of the wild-type CcO. The data indicate that the disabling of the proton pump is a result of a perturbation of E(I-286), which is located 20 A from N(I-139) and is an internal proton donor to the catalytic site, located in the membrane-spanning part of CcO. The mutation results in raising the effective pKa of E(I-286) by 1.6 pH units. An explanation of how the mutation uncouples catalytic turnover from proton pumping is offered, which suggests a mechanism by which CcO pumps protons.

Amino Acid Substitution↗